JP7142569B2 - Systems, methods and devices for sample collection, stabilization and storage - Google Patents

Description

RELATED APPLICATIONS [0001] This application claims priority to U.S. Provisional Patent Application No. 62/216,312, filed September 9, 2015, which is filed November 25, 2015. 62/260,172, which claims priority to U.S. Provisional Patent Application No. 62/367,056, filed July 26, 2016, filed July 29, 2016 No. 62/368,817, the entire disclosure of which is incorporated herein by reference.

[0002] Medical diagnostic tests can be life-saving, but unfortunately they are not readily available to everyone. Rising medical costs and availability of treatment can make it difficult for people around the world to obtain adequate treatment. Multiple visits to the doctor are required to get a simple blood panel test. First, to draw blood, then for follow-up consultations or additional tests. Substantial increases in health care costs could serve as an additional deterrent, with many patients unable to afford the time and expense of multiple visits to the doctor and testing until they experience symptoms that require treatment. may be postponed. New treatment paradigms can address patient needs by reducing the burden of time, cost, and availability. Improving patient access to diagnosis can increase the likelihood that testing will be performed before symptoms appear, allowing patients to receive treatment much earlier than previously possible. Early detection can facilitate early treatment, ensure a better prognosis for patients, and reduce the overall cost of treatment.

[0003] Provided herein is an improved blood sample that allows patients to collect, prepare, store, detect and analyze their own blood samples without the assistance of medical personnel or access to medical facilities. A solution is provided.

[0004] The present invention relates generally to medical systems, devices and methods, and more particularly to systems and methods for user collection, separation, and stabilization of blood samples. Specifically, the systems, devices and methods disclosed herein are one configured to collect blood from a subject when activated and stabilize the blood for further processing within or outside the device. An integrated device is provided that includes a sample acquisition component, a sample separation component and a blood stabilization component integrated within the somatic device.

[0005] These and other embodiments are described in greater detail in the following description in conjunction with the accompanying drawings.

[0006] In one aspect, a method is disclosed. The method includes positioning a sample acquisition and stabilization system in a subject with a tag, the system comprising a blood collection unit configured to collect a blood sample from the subject and a blood sample from the collected blood sample. an optional separation module for separating one or more components; and a stabilization matrix configured for stabilizing the one or more components from a collected blood sample, wherein the tag is Contains associated labels. The method further comprises collecting the deployed tagged sample acquisition and stabilization system, performing one or more assays on one or more stabilization components based on the tag, and performing providing a report on the one or more assays. The method may further comprise isolating the stabilization matrix from the system prior to one of the aforementioned method steps. A tag can be present on a stabilizing matrix. Optionally, if a separate module is present, the tag can be present on any separate module. The stabilization matrix can be configured to perform multiple assays. The method further comprises identifying a stabilized biological component within the blood sample based on the tag present in the deployed sample acquisition and stabilization system, and from that identification, the stabilization to be performed. determining the component-specific assays performed. The sample acquisition and stabilization system can be labeled with a second label different from the RFID transmitter, color label, bar code, or sample tag.

[0007] In another aspect, a method is disclosed for performing an analysis on one or more biological constituents from a biological sample of a subject. The method includes a sample acquisition component and one or more stabilization reagents configured to selectively stabilize at least one biological component selected from the group consisting of RNA, DNA and proteins. Obtaining a sampling device that includes a stabilization matrix. The method comprises performing one or more assays on at least one stabilized biological component in or on a sampling device remote from one of the above method steps. Including further. One or more stabilizing reagents can be disposed on or integrated with a solid matrix and are in a substantially dry state. The solid matrix can be configured to substantially stabilize RNA for at least 11 days at 37°C. Assays are performed in or on the device and may include one or more of PCR, DNA sequencing, RNA expression analysis, and RT-PCR.

[0008] In another aspect, a method is disclosed for performing an analysis on one or more biological constituents from a biological sample of a subject. The method comprises using one or more stabilization reagents configured to selectively stabilize a sample acquisition component and one or more biological components selected from the group consisting of RNA, DNA and proteins. Obtaining a sampling device comprising a stabilizing matrix comprising: The method further includes removing the stabilized matrix having one or more biological components from the sampling device and performing one or more assays on the stabilized matrix. The stabilization matrix can be retrieved from the sampling device at a location remote from performing one of the above method steps.

[0009] In another embodiment, a substantially dry composition comprising one or more stabilizing reagents that selectively stabilize one or more biological components selected from the group consisting of DNA, RNA and proteins. receiving a solid matrix under ambient conditions and performing one or more assays directly from the dry solid matrix to analyze one or more selectively stabilized biological components. and a method is disclosed. The stabilizing reagent can comprise a solid substrate comprising substantially dry melezitose having a water content of less than 2%. A stabilizing reagent can be attached to a sample acquisition component comprising a solid matrix for extracting and storing nucleic acids from a biological sample, the solid matrix having an RNA Integrity number (RIN) of at least 4.

[0010] In another aspect, a device for collecting and stabilizing a biological sample by point-of-care or self-administration is disclosed, and embodiments are illustrated herein. The device includes a sample acquisition component including a body and a plunger, the body having a proximal end, a distal end, an outer body surface extending between the proximal and distal ends, and at least one a base attached to the distal end; a lumen defined within the body surface; a plunger disposed within the lumen; With at least one needle connected to that end, the at least one needle is configured to pass through the at least one hole in the base when the plunger is actuated. The device includes sample stabilizing components, including reagents and substrates, that extract and stabilize components of the biological sample as it moves from the collection point through the matrix. The sample stabilizing component is disposed within the lumen of the body and actuating the plunger causes the biological sample to enter the matrix to further facilitate separation of the biological sample. becomes.

[0011] In another aspect, a kit for collecting and stabilizing a sample is disclosed. The kit includes a sample acquisition component having one or more piercing elements configured to penetrate the skin and expose blood, and a sample stabilizing component, the sample stabilizing component substantially A solid substrate containing melezitose in a dried state. A solid substrate can include one or more of a lysing reagent and a biomolecule stabilizing reagent. Optionally, melezitose can be present in concentrations ranging from 10-30%. Solid substrates can be configured to store enzymatic activity. The kit can further include a plasma separation component.

[0012] In another aspect, a kit for collecting and stabilizing a biological sample is disclosed. The kit includes a sample acquisition component having one or more piercing elements configured to penetrate the skin and expose blood, and one or more of biological components of RNA, DNA and protein. a sample stabilization component comprising a solid matrix for selective stabilization. A solid matrix can selectively stabilize RNA, and RNA obtained from a solid matrix can have a RIN value of greater than four. The kit can further include a plasma separation component. RNA obtained from the solid matrix can have a RIN of at least 5 or at least 6, or at least 7, or can have a RIN greater than 7. The solid matrix can further comprise at least one protein denaturant present in the solid matrix in its dry state. The solid matrix can further comprise at least one reducing agent in the solid matrix in the dry state. The solid matrix can further comprise at least one UV protection agent in the solid matrix in the dry state. The solid matrix can further comprise at least one buffer present in the solid matrix in its dry state. The buffer may be an acid titration buffer reagent that produces a pH in the range of 3-6. The solid matrix can be composed of cellulose, cellulose acetate, glass fibers, or combinations thereof. A solid matrix can be a porous matrix. A solid matrix may be an insoluble dry solid material. A solid matrix may be configured to provide an acidic pH upon hydration. A solid matrix can be configured to extract nucleic acids from a sample and store the nucleic acids in a substantially dry state at ambient temperature. A sample acquisition component can be connected to a sample stabilization component.

[0013] In another aspect, a system for collecting and stabilizing a sample is disclosed. The system includes a sample acquisition component having one or more piercing elements configured to penetrate the skin and expose blood, and a sample stabilization component coupled to the sample acquisition component, the sample The stabilizing component comprises a solid substrate comprising melezitose in a substantially dry state having a water content of less than 2%. A solid substrate can include a lysing reagent, one or more biomolecule stabilizing reagents, or a combination thereof. The concentration of melezitose can be present in the range of 10-30%. Solid substrates can be configured to store enzymatic activity.

[0014] In another aspect, a system for collecting and stabilizing a biological sample is disclosed. The system includes a sample acquisition component having one or more piercing elements configured to penetrate the skin to expose blood, and a sample stabilization component coupled to the sample acquisition component; A sample stabilizing component comprises a solid matrix for extracting and storing nucleic acids from a biological sample, wherein RNA obtained from the solid matrix has a RIN of at least 4. RNA obtained from solid matrices may have a RIN of at least 5, or at least 6, or at least 7, or may be greater than 7. The solid matrix can further comprise at least one protein denaturant present in the solid matrix in its dry state. The solid matrix can further comprise at least one reducing agent in the solid matrix in the dry state. The solid matrix can further comprise at least one UV protection agent in the solid matrix in the dry state. The solid matrix may further comprise at least one buffer disposed on or impregnated within the solid matrix, wherein the solid matrix is substantially dry and has a water content of 2%. is less than The buffer may be an acid titration buffer reagent that produces a pH in the range of 3-6. The solid matrix can be composed of cellulose, cellulose acetate, glass fibers, or combinations thereof. A solid matrix can also be a porous matrix. A solid matrix may be an insoluble dry solid material. The solid matrix can be configured to have an acidic pH upon hydration. A solid matrix can be configured to extract nucleic acids from a sample and store the nucleic acids in a substantially dry state at ambient temperature. A sample acquisition component can be connected to a sample stabilization component.

[0015] In another aspect, a system for collecting and stabilizing a biological sample is disclosed. The system includes a sample acquisition component having one or more piercing elements configured to penetrate the skin to expose blood, and a sample separation component coupled to the sample acquisition component, the sample acquisition component comprising: The components are configured such that one component of the biological sample is collected on the first membrane and the remainder of the sample is collected on the second membrane. Includes overlapping membranes.

[0016] In another aspect, a method is disclosed that includes positioning a tagged sample acquisition and stabilization system in a subject, the system comprising: a blood collection unit configured to collect a blood sample from the subject; , an optional separation module for separating one or more components from the collected blood sample; and a stabilization matrix configured for stabilizing the one or more components from the blood sample; The tagged sample acquisition and stabilization system contains a label associated with the assay. The method further comprises collecting the deployed tagged sample acquisition and stabilization system, performing one or more assays on one or more stabilization components based on the tag, and performing Providing a report on the one or more assays. The method may further comprise isolating the stabilization matrix from the system prior to one of the aforementioned method steps. Optionally, tags can be present on a stabilizing matrix. Optionally, if a separate module is present, the tag can be present on any separate module. The stabilization matrix can be configured to perform multiple assays. The method further includes identifying the stabilized biological component in the blood sample based on the tag present in the deployed sample acquisition and stabilization system, and the stabilized biological component to be derived from the identification. Determining a component-specific assay can include. The sample stabilization system can be labeled with a second label different from the RFID transmitter, color label, bar code, or sample tag.

[0017] In another aspect, a method is disclosed for performing an analysis of one or more biological constituents from a biological sample of a subject. The method includes a sample acquisition component and one or more stabilization reagents configured to selectively stabilize at least one biological component selected from the group consisting of RNA, DNA and proteins. Obtaining a sampling device that includes a stabilization matrix. The method further comprises performing one or more assays on the stabilized biological component in or on the sampling device at a location remote from where the first step above was performed. include. The one or more stabilizing reagents may be disposed on or integrated with the solid matrix and in a substantially dry state. The solid matrix can be configured to substantially stabilize RNA for at least 11 days at 37°C. Assays can be performed in or on the device and can include one or more of PCR, DNA sequencing, RNA expression analysis, and RT-PCR.

[0018] In another aspect, a method is disclosed for performing an analysis of one or more biological constituents from a biological sample of a subject. The method comprises using one or more stabilization reagents configured to selectively stabilize a sample acquisition component and one or more biological components selected from the group consisting of RNA, DNA and proteins. Obtaining a sampling device comprising a stabilizing matrix comprising: The method comprises removing a stabilized matrix having one or more biological components from a sampling device and performing one or more assays on or directly from the stabilized matrix. , further includes. The stabilization matrix can be retrieved from the sampling device at a location remote from one of the above method steps.

[0019] In another embodiment, a substantially dry composition comprising one or more stabilizing reagents that selectively stabilize one or more biological components selected from the group consisting of DNA, RNA and proteins. receiving a solid matrix under ambient conditions; and performing one or more assays directly from the dry solid matrix to selectively analyze the stabilized biological component. Disclose how. The stabilizing reagent can comprise a solid substrate comprising substantially dry melezitose having a water content of less than 2%. The stabilizing reagent can be bound to a sample acquisition component comprising a solid matrix for extracting and storing nucleic acids from a biological sample, wherein RNA obtained from the solid matrix has an RNA Integrity number (RIN ).

[0020] In another aspect, a device for collecting and stabilizing a sample by point-of-care or self-administration is disclosed, embodiments are illustrated herein. The device includes a sample acquisition component including a body and a plunger, the body having a proximal end, a distal end, an outer body surface extending between the proximal and distal ends, and at least one a bore, a base attached to the distal end, an inner surface defined within the body, a plunger disposed within the lumen, the plunger having a proximal end, a distal end, and/or a at least one needle connected to the end of the plunger, the at least one needle configured to pass through the at least one hole in the base when the plunger is actuated. The device includes sample stabilizing components, including reagents and substrates, that extract and stabilize components of the biological sample as it travels through the matrix from the collection point. The sample stabilizing component is disposed within the lumen of the body and actuation of the plunger causes the biological sample to enter the matrix to further facilitate separation of the biological sample. becomes.

[0021] In another aspect, a kit for collecting and stabilizing a sample is disclosed. The kit includes a sample acquisition component having one or more piercing elements configured to penetrate the skin and expose blood, and a sample stabilizing component, the sample stabilizing component substantially includes a solid substrate containing melezitose in a dry state. The solid substrate can further comprise one or more of a lysing reagent and a biomolecule stabilizing reagent. Melezitose can be present in concentrations ranging from 10-30%. Solid substrates can be configured to store enzymatic activity. The kit can further include a plasma separation component.
Incorporation by Reference

[0022] All publications, patents referred to herein to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. and patent applications are incorporated herein by reference.

[0023] Features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention may be had by reference to the following detailed description and accompanying drawings that illustrate illustrative embodiments in which the principles of the invention are employed.

[0024] FIG. 4 illustrates different sample acquisition components and lancing elements; FIG. 2 illustrates different sample acquisition components and lancing elements; FIG. 2 illustrates different sample acquisition components and lancing elements; [0025] FIG. 4 illustrates an embodiment of a sample acquisition component; [0026] FIG. 7 is a flow chart depicting a method for taking a blood sample using a tourniquet. [0027] FIG. 5 illustrates an embodiment of an isolation component. FIG. 13 illustrates an embodiment of the isolation component; [0028] FIG. 7 illustrates another embodiment of a sample separation component; [0029] FIG. 7 illustrates a sample stabilization component with a stabilization matrix; [0030] is a non-limiting list of tests that can be performed on a sample; A non-limiting list of tests that can be performed on a sample. A non-limiting list of tests that can be performed on a sample. A non-limiting list of tests that can be performed on a sample. A non-limiting list of tests that can be performed on a sample. A non-limiting list of tests that can be performed on a sample.

[0031] Statements

[0032] A first step toward improving the availability of biological sample testing and diagnostic testing is the rapid sample collection, stabilization and It may involve the development of systems and methods that enable preservation. Once stabilized, such biological components can be transferred to a laboratory where one or more biological assays can be performed on the biological component. Easier sample collection can be particularly beneficial for diagnosing clinical conditions. Collecting, preparing, and optionally analyzing a subject's own blood sample without the assistance of a physician and access to a medical facility, or collecting a biological sample from a subject by an untrained individual, Stabilizing may be beneficial. Technological developments in this area consist of at least three components: first, new developments to provide simple tools for collecting biological samples such as blood; New systems, devices and methods to provide simple and easy-to-use mechanisms for separating, stabilizing and/or preserving components, and new compatibility for analyzing samples provided via these new methods. A sensible approach can be used.

[0033] The new technology can be sufficiently easy to use and effective to enable sample acquisition, collection, optional separation and stabilization to be performed by trained and untrained end-users. Devices, systems and methods are disclosed that overcome current limitations by addressing some of the aforementioned problems.

[0034] The devices, systems and methods disclosed herein can provide an easier sample collection system that is easy to use for sample collection, optional separation, stabilization and sample storage. Additionally, the devices, systems and methods can provide approaches and tools for laboratories to more easily receive, prepare and analyze samples. Allows samples to be easily collected, stored, prepared and prepared for analysis so that sample detection can be easily accomplished with little effort on behalf of the user, patient or sample provider Provided herein is a compatible set of methods, tools and systems for The devices, systems and methods disclosed herein can reduce the burden of diagnostic testing by simplifying the process of biological sample collection, optional separation, and stabilization.

[0035] Simplifying the process for collection of blood samples can include devices, systems and methods for separating sample components. In some embodiments, the method for collecting venous blood comprises every step of the blood collection process, i.e., from collection of the sample, to separation steps including centrifugation, subsequent labeling and delivery of the sample to the laboratory for testing. One or more dedicated medical professionals may be relied upon to supervise the steps through post-collection procedures, which may include isolation steps, including refrigeration to stabilize until transported. In alternative embodiments, the systems and methods disclosed herein can be configured to allow end-users to self-manage blood sample collection and provide stabilized samples to detection facilities for analysis. can.

[0036] The systems, devices and methods presented herein are (i) for the simple collection of one or more biological samples (e.g., blood, urine, or environmental samples such as water or soil). (ii) one or more stabilization components for stabilizing biological analytes from one or more biological samples (e.g., DNA, RNA or protein); and (iii) an optional separation component (eg, plasma or cells) for separating one or more sample components. Additional components of the systems or methods disclosed herein can include one or more kits, devices, methods and systems for processing samples and analyzing user-provided samples.

[0037] Devices, systems, methods and kits include a sample acquisition component (SAC) for acquiring a sample and a sample stabilization component (SSC) for transporting, collecting and stabilizing the sample. can contain. Optionally, a sample stabilization component can further be provided to separate one or more components of the sample prior to transferring the sample to a solid substrate for stabilization. The SAC is easy to use and allows untrained professionals to collect samples at a variety of locations, from the doctor's office to the patient's home or office. SACs are dedicated providers that allow donors to collect their own samples at home without the need to visit a clinic, or sample collection can be done by trainers or untrained professionals. Allows collection at collection points.

[0038] Non-limiting embodiments can include integral and non-integral combinations of components for sample acquisition, sample separation, and sample stabilization. Embodiments can include a single integrated device with separate internal components for stabilizing the components of the sample. Additional integrated devices can include a single unit or two or more units for separating sample components prior to selectively stabilizing the sample components. Other embodiments may provide non-integrated components within the system or kit, the components being a sample acquisition component for acquiring the sample and a sample acquisition component for stabilizing and optionally separating the sample. and a separate sample collection component. The sample stabilizing component can include a solid stabilizing matrix for selectively stabilizing and preserving components of the sample, and can also have components for separating the sample prior to stabilization. In yet additional non-integrated systems or kits, the sample stabilization component can be separate from the sample separation unit. Further embodiments provide methods, devices, systems and methods for receiving, preparing, and/or treating a stabilized sample after the sample has been obtained, separated, and components of the sample have been selectively stabilized. Kits can be provided.

[0039] The terms "biological sample" and "biological sample" may be used interchangeably herein and throughout the specification. A biological sample can be blood or any effluent fluid. Non-limiting examples of biological samples can include saliva, blood, serum, cerebrospinal fluid, semen, feces, plasma, urine, cell suspensions, or suspensions of cells and viruses. In non-limiting examples, biological samples can include plant or fungal samples. The system and method can be applied to any biological sample from any organism, including humans. A biological sample obtained from an organism can be blood, serum, plasma, synovial fluid, urine, tissue or lymph. Biological samples include whole cells, lysed cells, plasma, red blood cells, skin cells, non-nucleic acids (e.g. proteins), nucleic acids (e.g. DNA/RNA), such as circulating tumor DNA (ctDNA) and cell-free DNA (cfDNA). ). Some embodiments may disclose methods, devices, and systems for collecting blood samples. However, the systems and methods disclosed herein are not intended to be limited to obtaining biological samples from organisms. For example, the disclosed embodiments can be used with samples obtained from the environment. Non-limiting examples of environmental samples include water, soil and air samples. In some cases, samples used in methods, compositions, kits, devices or systems provided herein are not biological samples.

[0040] For purposes of describing the devices, methods, systems, and kits disclosed herein, any individual who uses the device, method, system, or kit for collecting a sample is referred to as an "end user." can be called An individual, organism or environment from which a sample is derived may be referred to as a "donor." Once the sample is collected, it can be placed at another facility for testing. At the facility, the sample can undergo selected treatment steps based on the device, system, method or kit used.

[0041] Sample Acquisition Component (SAC)

[0042] The devices, systems, methods and kits described herein can include one or more sample acquisition components (SACs). Samples obtained by the systems provided herein can be biological samples of biological origin, e.g., blood, serum, urine, saliva, tissue, hair, skin cells, semen, or e.g., water samples, from wells samples obtained from the environment, such as the oil in the body, or oils from foods such as milk, and the like. A sample may be a liquid, a solid, or a combination of one or more liquids and one or more solids.

[0043] The sample volume may be the configuration of the unit including, but not limited to, the device collection chamber, the material properties of the collection system, the sample settings predetermined by the user, specifications established during device manufacture, or any combination thereof. Can be fixed by an element.

[0044] The SAC can include one or more devices for venous or capillary blood collection. To achieve venous or capillary blood sampling, the SAC can contain one or more piercing elements. The one or more piercing elements may be hollow or solid, and the one or more piercing elements may be configured for painless and efficient sample transfer. Adaptation can include the use of materials with specific compositions, surface microstructures, mechanical properties, structural geometries, or combinations thereof. The one or more piercing elements can include one or more needles, microneedles, or lancets (including pressure actuated needles or lancets). The SAC can optionally be designed to minimize physical pain or discomfort to the user. A SAC can include the microneedle technology shown in FIG. 1A. It can penetrate the skin shallowly to generate blood flow. Examples of sample, eg blood collection, contemplated herein include those described in US Pat. No. 9,033,898. The SAC may be single use. In some cases, the SAC is reusable.

[0045] The SAC can, for example, collect volumes of less than 1 mL. For example, the SAC can be configured to collect blood volumes of 1 ml, 500 μl, 400 μl, 300 μl, 200 μl, 100 μl, 90 μl, 80 μl, 70 μl, 60 μl, 50 μl, 40 μl, 30 μl, 20 μl or 10 μl.

[0046] The SAC can be designed to collect a sample volume of about 1 mL to about 10 mL or about 1 mL to about 100 mL. Examples of SACs include urine cups, finger sticks, or devices such as those described in US Patent Application Publication Nos. 2013/0211289 and 2015/0211967.

[0047] The SAC can be a component of an integrated device designed for sample collection, stabilization and storage. The SAC may be a separate component that is part of a kit or system. The SAC can include vials for collecting samples.

[0048] A kit can include one or more of the following (eg, where the SAC is non-integrated but part of the kit). (i) one or more sample separation units, (ii) one or more sample stabilization units, (iii) one or more biological sample separation and/or stabilization components. Kits used for blood samples collect a drop of blood from a pricked or incised finger and subsequently transfer the blood to a device or separately for stabilization or to separate the stabilized sample components. can be provided with capillaries or transfer tubes for distribution to the units.

[0049] Some embodiments of sample acquisition components are illustrated in FIGS. As shown in FIG. 1B, the SAC can be actuated through mechanical means. Manual activation may be performed by the donor or end user. FIG. 1C illustrates a SAC that can be actuated by electrical means. As shown, the SAC can use vibration to induce blood flow.

[0050] The SAC uses a plunger to create a vacuum that can force a sample into one or more chambers within the device. As shown in FIG. 2, the sample acquisition component (SAC) can have a proximal end, a distal end, an outer surface, and a lumen defined by body 215 . A base attached to the distal end of the body can include an outer surface, an inner surface, and can include one or more holes 240 . Additionally, the device can have a plunger 220 that includes a proximal end and a distal end, the plunger being configured for user actuation. One or more piercing elements 250 can be secured to the face of the plunger, and can pierce the skin as the user actuates the SAC from the proximal end to the distal end of the device body. A vacuum may be created upon withdrawal of the piercing element. Blood can be pooled through one or more holes 240, and one or more holes can be configured to draw the sample into the lumen. Samples can move within lumens and devices, for example, by naturally occurring capillary forces. Naturally occurring capillary forces can also be used to extract fluids into reservoirs from droplets on surfaces such as pools or human skin, or from other open microfluidic channels. Naturally occurring capillary forces can be used to collect samples from the incision site, as well as sample collection and preparation steps from reservoirs within the device or using complex open microfluidic channels. It can also be used to manipulate fluids in between. In alternative embodiments, spontaneous capillary-driven flow can be combined with other means of moving the sample through the device. The aperture can optionally be adapted to or include a material or structure suitable for drawing sample into the device from a punctured skin. For example, in one embodiment, one or more piercing elements can be partially retracted into one or more holes, and properties of the one or more piercing elements themselves cause the one or more piercing elements to be hollow. Sample chamber via a hole, either through the one or more piercing elements in some cases, or on the side of the one or more piercing elements if the one or more piercing elements are solid. Bring the sample inside. Embodiments of these components can be found in US Patent Publication No. 2013/0211289.

[0051] Sample collection can be from samples pooled at or above the skin surface, optionally from one or more reservoirs under the skin. The SAC can produce a lancing motion that cuts small but abundant capillaries in the subepidermal superficial vascular plexus, eg, at a depth of 0.3-0.6 mm from the skin surface. The present disclosure provides systems for mechanically massaging an incision site at other body locations by several different approaches, including needles or capillary tubes or vibrating paddles, or applying desired blood flow. Vibrating an annular ring surrounding the wound to pump blood surrounding the wound into the wound, and the like, for sampling by another member adjacent the wound to achieve. In addition, it can be difficult to carry a drop of blood from other body areas, such as the skin of the thigh, to a small area on the testing device. Alternative embodiments described herein can use a needle that remains within the wound, and the needle can be mechanically manipulated to facilitate formation of a fluid sample within the wound.

[0052] A liquid sample can be collected or pooled in a collection chamber, where porosity and absorbency are optimized to draw the sample into the device after, or instead of, the collection chamber. can optionally be absorbed through one or more particles, materials, structures or filters. Materials for drawing samples into the devices herein can consist of any material with an absorbent or adsorbent surface, or modified surface, including, but not limited to, paper-based media, gels, beads, membranes, polymer-based matrices, or any combination thereof; For example, in one embodiment, the SAC can comprise a body that defines a fluid flow path from the inlet opening, the flow path extending through the inlet opening and the matrix to be dispensed or dispensed through the bed. It includes a bed of porous polymeric monoliths selected to adsorb biological particles or analytes. Porous polymer monoliths can absorb biological particles or analytes for later preparation steps. An example of sample collection on a porous monolith can be found in US Patent Application Publication No. 2015/0211967.

[0053] A method for using a sample acquisition component (SAC) may include piercing the skin with the SAC after milking or squeezing a finger to obtain a blood sample. The SAC can be used with tourniquets or other components to facilitate sample acquisition. A tourniquet, rubber band, or elastic material can be placed around the first, second, or third finger of the subject's hand. Methods can be constructed to improve sample quality. As depicted in FIG. 3, the steps may include placing 305 a tourniquet on one of the donor fingers and applying pressure, and cutting 310 the finger to form an incision. The method may also optionally include drawing the first blood immediately after the puncture under pressure. Blood can be collected 315 from the incision by holding the capillary tube against the blood droplet formed from the incision site. Blood collected in capillary tubes can be dispensed 320 onto separate components of the system, and separate portions of the components can be placed 325 at different locations or facilities for analysis. In certain embodiments, the kit may include components and instructions to facilitate blood collection and efficiency. The method further includes preparing the hand and fingers to ensure proper blood flow, including temperature and position, applying a tourniquet to the finger, sterilization, puncture, and the actual blood collection method. be able to. Methods of collecting blood samples are disclosed, for example, in US patent application Ser. No. 14/450,585.

[0054] Sample stabilization component

[0055] A sample acquisition component, eg, a sample acquired using a SAC described herein, can be transferred to a sample stabilization component (SSC). A SSC is any device or system on which samples are collected or transported for stabilization and storage. A device or system can include channels and compartments for collecting and transporting samples, and one or more units for separating and stabilizing samples.

[0056] In a non-integrated system, the sample acquisition component can be physically separated from the sample stabilization component. In these embodiments, transfer from the sample acquisition component to the sample stabilization component is via various means such as one or more needles, capillary tubes, or from the donor site to the sample stabilization component. can be done via direct transfer of If the sample acquisition component is a separate component from the sample stabilization component, the application of the sample to the substrate uses a self-filling capillary to collect from the sample acquisition component, after which the sample is transferred to the substrate. can be achieved by moving to

[0057] The sample stabilization component can be integrated with the SAC. Integration between the sample stabilization component and the SAC can be done through a shared assembled interface. The sample may be directed through a channel, including microchannels, through naturally occurring capillary forces, wicking through an absorbent material, or with minimal effort on behalf of the end user, through a sample stabilization component, or onto a substrate. It can be moved from the sample acquisition component, such as through other means that can flow into the substrate. Microchannels are constructed from a variety of materials that have properties including adapted shapes with surface microstructures and material properties adapted to promote capillary action or other means of transporting samples through the device. can do. Microchannels can include any means of transferring samples between chambers, such as open microfluidic channels optimized to move samples using naturally occurring capillary forces. Examples of microchannels and devices containing microchannels can be found in many of the incorporated references, such as US Patent Application Publication No. 2014/0038306.

[0058] The sample stabilization component can include a structure having multiple layers. It can also have an interface to allow transfer to one or more layers or substrates. A sample stabilizing component can be configured to collect one or more samples, separate sample components, stabilize one or more samples, or any combination thereof. SSCs are layers and capillaries configured to transfer a sample into a substrate between multiple layers, for example, as described in U.S. Patent Application Nos. 14/340,693 and 14/341,074. It can further include a network of channels.

[0059] The SSC can be attached to the substrate in a variety of ways. The SSC can be attached to the substrate in a manner that allows contact with the layer to transfer sample fluid from the integrated device to the substrate. The SSC can be configured to allow easy removal of the device from the substrate. The system can further include a substrate frame having an area configured to receive the sample on the substrate. The substrate can be attached to a substrate frame so that the substrate can be easily removed from the system, and the substrate frame can be designed with barcodes to allow for automated or semi-automated processing. The system can further be coupled to external devices, including fluidic devices, analytical instruments, or both. In one or more embodiments, the sample stabilization component can be coupled to the substrate, and the SSC is configured to transfer sample fluid to the substrate. A substrate can include a substrate or a sample separation component. The SSC can be attached directly to either the substrate or the substrate frame that holds the substrate. In some embodiments, the SSC can be further coupled to the substrate frame and substrate cover. The substrate frame and substrate cover can include features that facilitate efficient fluid transfer to the substrate at the area of interest, eg, the center of the substrate. In some embodiments, the SSC is a substrate that is packaged with a sample storage substrate pre-attached with a sample stabilization component. In some other embodiments, the SSC and substrate are packaged separately and the user can assemble the substrate and SSC for sample collection, transport, stabilization and storage. The SSC can be further packaged with a sample acquisition component (SAC).

[0060] SSCs are disposable or reusable. For example, the SSC may be a single-use, disposable device configured to collect a sample, transfer a sample fluid to a substrate, and facilitate loading of the fluid sample through a desired area of the substrate. . The SSC can be configured for single use to reduce or prevent contamination or spread of infection through collected samples. SSCs can be configured for reliable and reproducible collection, transfer and storage of biological samples.

[0061] After collection and transfer of the biological sample, the substrate can be configured to separate the biological sample components prior to transfer to a stabilizing matrix for storage.

[0062] Sample Separation

[0063] The SSC can include a sample separation unit that includes one or more substrates, membranes, or filters for separating sample components. The sample separation unit can be integrated within the sample stabilizing component, attached to the sample stabilizing component, or separate from the sample stabilizing component.

[0064] Sample separation can occur at different points in the sample collection process. For example, in an integrated device, sample separation may occur within the SSC, and in a non-integrated device, sample separation may occur outside the SSC prior to transfer to the sample stabilization component. In another example, moving the sample through the SAC to the sample separation unit and then to the SSC where the separated sample can be transferred to one or more substrates for stabilization and storage. can be done.

[0065] Sample separation can be performed as an intermediate step between sample acquisition and transfer to a sample stabilization matrix. In some cases, sample separation and stabilization can occur in one step without requiring user intervention. Separation of samples can also be performed sequentially or simultaneously with sample stabilization.

[0066] Sample acquisition and stabilization may require user action to progress through one or more stages of the sample collection, optional separation, and stabilization process. Integrated devices may require user action to effect sample acquisition and to move samples during separation, stabilization, and storage. Alternatively, user action may be required to initiate sample acquisition and one or more additional steps of the sample collection, separation or stabilization process. User actions include pressing buttons, tapping, shaking, rupturing internal parts, twisting or turning components of the device, pushing samples into one or more chambers and any number of other mechanisms. , can list any number of acts. Transfers to these stages may occur in parallel with sample collection or may occur after sample collection. At any time during or prior to the processing step, the entire sample or components of the sample can be subjected to any number of techniques or treatment strategies for pretreatment of the cells of the biological constituents of the sample, including possible treatments. These include, but are not limited to, treatment with reagents, detergents, evaporative techniques, mechanical stress or any combination thereof.

[0067] The devices, methods, systems and kits disclosed herein can include one or more sample separation units. The sample separation unit can be used, for example, to separate plasma from blood, cells from water samples, or cells from acellular components. For blood samples, one or more components can be used to separate plasma or specific cells from other components of the blood sample. Alternatively, the separation devices, methods and systems selectively separate any number of sample components such as cells, plasma, platelets, specific cell types, DNA, RNA, proteins, inorganic materials, drugs, or other components. can be separated into

[0068] Non-limiting embodiments of the sample stabilization unit can also use the sample separation component to separate other non-plasma components. A sample separation component may be a sample acquisition component, such as through one or more channels including one or more microchannels, wicking of an absorbent material, or other means by which sample may flow through the device. can be connected to Systems and methods for separating samples are exemplary and non-limiting.

[0069] There can be many ways to make the separation, some of which use size, deformability, shape or any combination thereof. Separation can occur via one or more membranes, chambers, filters, polymers, or other materials. Membranes, substrates, filters and other components of the device may be chemically treated to selectively stabilize components, facilitate sample flow, dry samples, or any combination thereof. can be treated. Alternative separation mechanisms may include liquid-liquid extraction, solid-liquid extraction, and selective precipitation of target or non-target elements, charge separation, binding affinity, or any combination thereof. A separation stage may include one or more steps, each step relying on a different mechanism to separate the sample. One such mechanism can utilize size, shape or deformation to separate larger components from smaller components. Cell separation can be performed, for example, via a sorting device that can rely on one or more filters or other size exclusion methods to separate the components of the sample. Separation can also be achieved through selective binding, where specific components are separated by a binding event and unbound eluate migrates to or through an alternate chamber.

[0070] In some methods, a single membrane can be used to separate and collect one or more sample components from a bulk sample. Single-membrane methods can use a device that allows a sample to be applied to one end of the membrane, and as the sample flows through the first component of the sample, e.g. Based on this, it can be separated from the second component of the sample, eg plasma. After manipulation of the device, the first component of the sample, in this example the membrane containing the cells, can be cut from the second component of the sample, in this example the portion containing the plasma, cutting the membrane. requires the process of Alternatively, two separate membranes may be used, specifically a first membrane for separating one component, e.g. blood cells, and a second one for collecting the other component, e.g. plasma. Two membranes can be used for separation and collection of sample components. These membranes separate large components, e.g. cells, through the first membrane and the may be positioned to facilitate collection of a second, smaller component, such as plasma, via.

[0071] FIGS. 4A-B illustrate a sample separation unit that can be used to separate samples before or in parallel with stabilization. A sample separation unit can comprise a frame 400 , a separation membrane 452 and a collection membrane 454 . The frame can include an inner side positioned proximate to the first peripheral portion of the frame. Interior 402 is formed from a plurality of first slots in frame 416 . The frame further includes an outer side 404 arranged to surround at least a portion of the plurality of first slots 416 . Outer side 404 is formed from a plurality of second slots in frame 432 . The distal end of the separation membrane 420 is positioned on the outside and the proximal end of the collection membrane is positioned below at least one of the outside and the inside such that the proximal end of the collection membrane is aligned with the distal end of the separation membrane. has an overlapping contact area 428 with the . Further, the outside is configured to apply pressure to the separation membrane and the collection membrane around the overlapping contact area. Frame 400, separation membrane 452, and collection membrane 454 can be constructed of different materials. The frame 400 can be constructed of polymeric materials such as polypropylene, nylon (polyamide), high density polyethylene (HDPE), and polyetheretherketone (PEEK), and the frame can be manufactured using injection molding techniques. and have a uniform thickness. Separation membrane 452 can comprise a suitable material such as cellulose, fiberglass, cellulose acetate, polyvinylpyrrolidone, polysulfone, polyethersulfone, polyester, or combinations of these materials, and the separation membrane can conform to the geometry of frame 400. It can be designed to have a shape, specifically a geometry that matches the geometry of the inner side 402 of the frame 400 . Collection membrane 454 may comprise suitable materials such as cellulose, fiberglass, cellulose acetate, polyvinylpyrrolidone, polysulfone, polyethersulfone, polyester, or combinations of these materials, and the collection membrane may be chemically treated. . Another embodiment and method is to insert the distal end 458 of isolation membrane 452 under second distal end portion 436 on the outside of frame 404 via first central slot 416b on inside 402. , such as by pushing the inside 402 of the frame downward 460 . The method further includes displacing the outer side 404 and the inner side 402 by applying pressure, for example by pushing upwardly 466, through a second intermediate slot in the outer side 432b and the proximal end of the collection membrane 454. 462 can be inserted under at least one of the outer and inner sides such that the proximal end of the collection membrane has an overlapping contact area 468 with the distal end 458 of separation membrane 452 .

[0072] The separation mechanism may be optional, for example, it may be part of a modular system that allows the user or manufacturer to insert the cartridge into the sample path. In one possible embodiment, the sample can be transferred from any of the aforementioned collection devices to the secondary chamber. Transfer can be facilitated by user action or can occur spontaneously without user interaction.

[0073] The sample separation unit can separate sample components using filtration membranes. Figure 5 illustrates a sample separation unit having a filtration membrane that separates the non-cellular fraction of a biological sample. Filtration membrane 502 can have a sample application zone 510 and a transport zone 512 . The filtration membrane can be in direct contact with solid matrix 504 via transfer zone 512 . A biological sample can be applied to the sample application zone 510 of the filtration membrane and filtered as it travels through the filtration membrane. The filtration membrane can have multiple pores. Once the biological sample has passed through the filtration membrane, the resident intact cells within the biological sample, for example, can be mostly retained by the filtration membrane in the sample application zone 510, and the non-cellular fraction can be It can be filtered through the pores until it reaches transfer zone 512 and transferred and collected on a dry solid matrix. The filtration membrane can have pore sizes that can range from about 0.01 microns to about 5 microns. Filtration membranes can have pore sizes ranging, for example, from about 1 micron to about 2 microns. Pore size can vary from about 0.22 microns to about 2 microns. If a 1 micron pore size filtration membrane is used, any other circulating eukaryotic and/or pathogenic cells with a diameter greater than 1 micron can be retained within the filtration membrane and thus adhere to the dry solid matrix during filtration. never reach. Additional isolation components are described in US Patent Application Publication No. 2015/0031035.

[0074] Filtration can occur at various points within the sample collection process. The non-cellular fraction of the sample can be filtered from the biological sample at its own collection point, for example. Filtration can be performed without any prior pretreatment of the biological sample. Additionally, filtration can be performed in the absence of any stabilizing reagents.

[0075] The filtration membrane can be made from a variety of materials. The materials used to form the filtration membrane can be natural, synthetic, or synthetically modified naturally occurring materials. Suitable materials that can be used to make the filtration membrane include, but are not limited to, glass fiber, polyvinyl alcohol-bonded glass fiber, polyethersulfone, polypropylene, polyvinylidene fluoride, polycarbonate, cellulose acetate, nitrocellulose, hydrophilic foamed poly(tetrafluoroethylene), anodized aluminum, track-etched polycarbonate, electrospun nanofibers or polyvinylpyrrolidone. In one example, the filtration membrane is formed from a polyvinyl alcohol-bonded glass fiber filter (MF1™ membrane, GE Healthcare). In another example, the filtration membrane is formed from asymmetric polyethersulfone (Vivid™, Pall Corporation). In some embodiments, filtration membranes can be formed by a combination of two or more different polymers. For example, the filtration membrane can be formed by a combination of polyethersulfone and polyvinylpyrrolidone (Primecare™, iPOC).

[0076] After filtration, the separated non-cellular fraction can be collected on a dry solid matrix by physical interaction. Non-cellular fractions can be collected on dry solid matrices by adsorption or absorption.

[0077] Stabilization matrix

[0078] SSCs can be used to transport, stabilize and preserve target components of biological samples, which can include target components such as nucleic acids, proteins, and their respective fragments. SSCs can receive, extract, and stabilize one or more of these analytes on a substrate that can be bound to or contained within a sample stabilization component. FIG. 6 illustrates an example of a sample stabilizing component 610 having a sample substrate or matrix 615 for storing or stabilizing the sample. Substrate 615 can be held in place on one side of the device using frame 620 . Samples can be collected and transferred through channel 625 to an attached sample substrate.

[0079] As used herein, the term "target component" can refer to one or more molecules, eg, biomolecules, that can be detected or examined in a given diagnostic test. Target components for a particular test may need to be properly stored and stabilized for good quality diagnostic results.

[0080] The nature of the sample may depend, for example, on the source of the material, eg, biological material. For example, sources can be from a range of organisms including, but not limited to, viruses, bacteria, plants and animals. The source can be a mammalian or human subject. For mammalian and human sources, the sample may be selected from the group consisting of tissue, cells, blood, plasma, saliva and urine. In another aspect, the biological sample is selected from the group consisting of biomolecules, synthetically derived biomolecules, cell components and biologics.

[0081] Since the composition, stability and quality of target constituents can vary depending on their source, various Different substrates can be used. The chemistry and properties of the target component may vary depending on the origin of the sample, and the required degree of sample stabilization may depend on the intended diagnostic panel of samples. For example, some panels may require high concentration and low quality samples, while other panels may require low concentration and high quality samples. High quality reproducible test results may require high quality samples. The composition of the substrate as well as the substrate and method for stabilizing the sample can vary between assays and between target components or analytes.

[0082] The substrate or matrix composition may also be selected or configured for a particular target component, as the properties of the target component may vary. The intended sample source and/or diagnostic test for a particular sample can be variables that determine the composition of the substrate or matrix. For example, high quality diagnostic test results may require high quality or effective stabilization of the target component. In some cases, the composition of the substrate or stabilization matrix can be designed to specifically stabilize particular target components (eg, DNA, RNA or proteins). The matrix can be further configured for use in a particular diagnostic test, e.g., if the test requires a higher concentration of a particular type of target component, the matrix selectively targets that target component. Can be designed to emit

[0083] Sample processing can be performed keeping in mind that test and substrate composition types are different. For example, a substrate or matrix designed for a particular target component or diagnostic test can be indicated with a color or code that indicates the type of assay in which it can be used or with which it can be used. The substrate can also incorporate one or more tags or labels, such as barcodes, RFIDs, or other identifiers, that can tie samples or results derived from the substrate to a particular end-user or donor.

[0084] The term "substrate" or "stabilization matrix" can refer to any solid matrix, such as a substrate or sample separation component described herein. The substrate can be any solid material containing one or more absorbent materials capable of absorbing a fluid sample such as blood.

[0085] The substrate or stabilization matrix can comprise one or more different solid components. The solid component can be kept substantially dry with less than 10% hydration by weight. Examples of solid matrix substrates include, but are not limited to, naturally occurring materials, synthetic materials, or synthetically modified naturally occurring materials. Substrates include cellulose, nitrocellulose, modified porous nitrocellulose or cellulosic substrates, polyethylene glycol modified nitrocellulose, cellulose acetate membranes, nitrocellulose mixed ester membranes, glass fibers, polyethersulfone membranes, nylon membranes, polyolefin membranes, polyester membranes. , polycarbonate film, polypropylene film, polyvinylidene fluoride film, polyethylene film, polystyrene film, polyurethane film, polyphenylene oxide film, poly(tetrafluoroethylene-co-hexafluoropropylene) film, glass fiber film, quartz fiber film or combinations thereof can include Suitable materials that can act as a dry solid matrix include, but are not limited to, cellulose, cellulose acetate, nitrocellulose, carboxymethylcellulose, quartz fibers, hydrophilic polymers, polytetrafluoroethylene, glass fibers and porous ceramics. be done. Hydrophilic polymers may be polyesters, polyamides or carbohydrate polymers.

[0086] The substrate or matrix can include one or more dry reagents impregnated therein. The one or more dry reagents may include protein stabilizing reagents, nucleic acid stabilizing reagents, cell lysing reagents, or combinations thereof. In one embodiment, the substrate is placed on a substrate frame. Non-limiting examples of sample substrates can include porous sample substrates, Whatman FTA™ cards, cellulose cards, or combinations thereof. In some embodiments, the substrate can include at least one stabilizing reagent that preserves at least one biological sample analyte for transport or storage. Non-limiting examples of suitable reagents for the storage medium include weak bases, chelating agents, and optionally additives of one or more of uric acid or urates or chaotropic salts alone or in combination with surfactants. can be mentioned.

[0087] In some cases, the substrate can comprise a dry solid matrix comprising cellulose. Cellulosic dry solid matrices do not contain any detergents. Cellulosic dry solid matrices cannot be impregnated with any reagents. Cellulosic dry solid matrices can be impregnated with chaotropic salts. Examples of chaotropic salts include, but are not limited to, guanidine thiocyanate, guanidine chloride, guanidine hydrochloride, guanidine isothiocyanate, sodium thiocyanate, and sodium iodide. In some embodiments, the cellulosic dry solid matrix is FTA™ Elute (GE Healthcare). Examples of sample stabilization components can be found, for example, in US Patent Application Publication Nos. 2013/0289265 and 2013/0289257.

[0088] The substrate may be composed of one or more layers of material. Substrates can be aligned within a solid matrix. Layers can be aligned to selectively extract specific biological sample components. A solid matrix may be a single material or may comprise multiple materials.

[0089] Multiple sample stabilization units can be stored or attached to a single sample stabilization component. A single sample stabilization unit can be stored in the sample stabilization component. Alternatively, two or more sample stabilization units can be linked together, thereby allowing one or more components of the biological sample to move through different units. The sample stabilization matrix or substrate in any of the above embodiments can have a uniform or variable composition. Substrates, sample stabilization units, or components of either the substrate or the sample stabilization unit may be of the type of target component(s) for which they are intended and/or the diagnostic test(s) for which the sample is intended. can be tagged to indicate

[0090] The substrate may be constructed such that sample processing occurs within or near the sample stabilization component. In some embodiments, the sample can move through a sample separation step before being subjected to one or more sample stabilizing matrices. In some embodiments, the acquisition step, optional separation step and stabilization step are performed in parallel.

[0091] The substrate can be configured to enhance recovery for target components present at low concentrations. In these cases, the solid substrate can include at least one surface coated with a chemical mixture that enhances recovery of biological material from the surface. Chemical mixtures include vinyl polymers and nonionic detergents, vinyl polymers and proteins, synthetic nonionic polymers and nonionic detergents, synthetic nonionic polymers and proteins, polyethyleneimine (PEI) and nonionic detergents. , nonionic detergents and proteins, and polyethylenemines (PEI) and proteins. Solid supports may be selected from the group consisting of paper, glass microfibers and membranes. The support can be, for example, paper such as cellulose paper such as 903 Neonatal STD card. A solid support may comprise a membrane selected from the group consisting of polyester, polyethersulfone (PES), polyamide (nylon), polypropylene, polytetrafluoroethylene (PTFE), polycarbonate, cellulose nitrate, cellulose acetate and aluminum oxide. . The vinyl polymer can be polyvinylpyrrolidone (PVP). A non-ionic detergent can be Tween20. Proteins can be selected from the group consisting of albumin and casein. The nonionic synthetic polymer can be poly-2-ethyl-2-oxazoline (PEOX). The chemical mixture can include any combination of polyvinylpyrrolidone (PVP), Tween 20, albumin, poly-2-ethyl-2-oxazoline (PEOX), polyethyleneemine (PEI), polyethyleneemine (PEI) or casein. . Also herein, i) contacting a sample comprising a biological material with, for example, the surface of a solid support as described herein, ii) drying the sample on the surface of the solid support, Also provided is a method for recovering material, e.g., biological material from a solid support, comprising iii) storing the solid support, and iv) extracting the material, e.g., biological material from the surface. be done. In another aspect, step iii) comprises storing the paper support at a temperature in the range of about 15°C to about 40°C. Paper supports can be stored at lower temperatures depending on the thermal stability of the biological material. A method of making a substrate can include coating at least one surface of the support with a chemical mixture solution that enhances recovery of biological material from the surface, the chemical mixture comprising polyvinylpyrrolidone (PVP) and Tween20, polyvinylpyrrolidone (PVP) and albumin, Tween20 and albumin, poly-2-ethyl-2-oxazoline (PEOX) and Tween20, poly-2-ethyl-2-oxazoline PEOX and albumin, polyethyleneemine (PEI) and Tween20 , and mixtures selected from the group consisting of polyethyleneemine (PEI) and albumin. A sample stabilizing component can provide the use of solid supports as described herein to enhance the recovery of material, e.g., biological material, from its surface, and the disclosed stabilizing Components and methods are described, for example, in US Patent Publication Nos. 2013/0323723 and 2013/330750, which are incorporated herein by reference in their entireties.

[0092] A substrate or solid matrix can be configured to selectively preserve nucleic acids such as RNA, DNA and fragments thereof. A solid matrix for selectively stabilizing nucleic acids may comprise at least one protein denaturant and at least one acid or acid titration buffer reagent impregnated and stored dry. The matrix can be configured to provide an acidic pH upon hydration, extract nucleic acids from a sample, and/or store nucleic acids in a substantially dry state at ambient temperature.

[0093] Stabilizing matrices for extracting and stabilizing nucleic acids include, in a solid matrix, in a dry fashion, an agent such as a protein denaturant, a reducing agent, a buffer, a free radical trap, a chaotropic agent, a detergent, or an RNase inhibitor. Any combination of reagents can be included. RNase inhibitors can include triphosphates, pyrophosphates, acids, or acid titration buffers. The stabilizing matrix can be further impregnated with or in the presence of one or more reagents such as enzyme inhibitors, free radical scavengers, or chelating agents. Solid matrices can include protein denaturants, reducing agents, buffers, and optionally free radical traps or RNase inhibitors.

[0094] It can be impregnated with one or more reagents and stored dry. One or more dried reagents can optionally be rehydrated by addition of buffer, water or sample. The matrix can further include weak or strong protein denaturants. In certain embodiments, the solid matrix is porous cellulosic paper such as commercially available 903, 31-ETF, or FTA Elute™. The performance of this method makes it possible to store nucleic acids, eg RNA, which can be labile biomolecules, under ambient temperature in a dry manner (eg on a solid matrix). A solid matrix can be configured such that hydration of the matrix results in an acidic pH. As noted above, in one or more embodiments, RNA quality can be determined by capillary electrophoresis of extracted RNA through a biological analyzer. A dry solid matrix can allow long-term storage of one or more biological components, including nucleic acids (eg, RNA, DNA), in a dry manner under ambient conditions. In another aspect, the dry solid matrix for ambient extraction and storage of nucleic acids (e.g., RNA, DNA) from a sample comprises a thiocyanate salt, a reducing agent, a buffer, and optionally a thiocyanate salt present in the solid matrix in a dry fashion. Contains free radical traps or RNase inhibitors. A dry solid matrix for the extraction and storage of nucleic acids (e.g., RNA, DNA) from a sample comprises at least one metal thiocyanate, wherein the at least one metal thiocyanate is guanidinium thiocyanate (GuSCN) , reducing agents, buffering agents, and optionally not free radical traps or RNase inhibitors. The solid matrix may contain nucleic acids (e.g., RNA, DNA) in dry form and may be subjected to a process to release the nucleic acids from the solid matrix in an intact manner suitable for further analysis of the collected nucleic acid sample. .

[0095] RNA quality can be quantified as a RIN value, which can be calculated by algorithmic evaluation of the amounts of various RNAs present within the extracted RNA. High quality cellular RNA can exhibit RIN values approaching ten. In one or more embodiments, RNA extracted from the dried matrix has a RIN value of at least 4. In some embodiments, the matrix provides for ambient extraction and stabilization of biological samples, producing intact, high quality RNA with RIN values ranging from about 4 to about 10, or some In embodiments, the RIN value ranges from about 5 to about 8. The matrix may be a porous, non-dissolving dry material configured to provide a pH of about 2 to about 7 upon hydration for extracting RNA. This matrix is capable of stabilizing the extracted RNA and has an RNA Integrity number (RIN) of at least four.

[0096] A method for extracting and preserving nucleic acids from a sample comprises providing a sample to a dry solid matrix comprising a protein denaturant and an acid or acid titration buffer reagent; Occasionally, the step of producing an acidic pH, drying the matrix containing the extracted nucleic acid, and storing the extracted nucleic acid on the matrix substantially dry at ambient temperature can be included. Examples of the aforementioned sample stabilization components can be found in US Patent Application Publication No. 2013/0338351.

[0097] The sample stabilization unit can include a solid matrix for collecting and stabilizing non-nucleic acid components, such as proteins. In these examples, the substrate can be configured to extract proteins or peptides from a biological sample and stabilize them for storage at ambient temperature. In one such embodiment, a solid substrate for biomolecule collection, stabilization and elution can comprise a substantially dry trisaccharide. Trisaccharides can be selected from melezitose, raffinose, maltotriulose, isomaltotriose, nigerotriose, maltotriose, ketoses or combinations thereof. One or more embodiments of the solid substrate can further comprise melezitose in a substantially dry state. Melezitose can be a non-reducing trisaccharide sugar with a molecular weight of 504.44 g/mol. In one or more embodiments, the solid substrate may comprise melezitose, and the concentration of melezitose is in an amount less than 30%. Melezitose can be impregnated into the substrate. Substrates may also be passively coated or covalently modified with melezitose. In one or more examples, the substrate is further impregnated with one or more reagents, such as one or more lysis reagents, one or more buffering reagents or one or more reducing agents. In some embodiments, the one or more impregnating reagents are biomolecular stabilizing reagents such as cell lysing reagents, protein stabilizing reagents impregnated therein in a substantially dry state, protein storage chemicals, and combinations thereof. contains oxidizing reagents. Examples of the above systems and other embodiments are disclosed in US Patent Application Publication No. 2014/0294942, which is incorporated by reference in its entirety.

[0098] Other substrates for stabilizing proteins can include solid paper-based matrices comprising cellulose and/or glass fibers and hydrophilic or water-soluble branched carbohydrate polymers. The surface weight of the solid-based matrix can be 40-800 g/m ² . The hydrophilic or water-soluble branched carbohydrate polymer can be 4-30% by weight of the matrix. A hydrophilic or water-soluble branched carbohydrate polymer may have an average molecular weight of 15-800 kDa, such as 20-500 kDa. Branched carbohydrate polymers may include dextrans. The dextran may be a branched (16) linked glucan. Branched carbohydrate polymers can include copolymers of mono- or disaccharides and difunctional epoxide reagents. Such polymers can be highly branched due to the multiple reactive hydroxyl groups on each mono/disaccharide. Depending on the reaction conditions used, the degree of branching can be from about 0.2 to nearly one. The water extract content in the paper may be 0-25% by weight, for example 0.1-5% or 3-20% by weight. Very little extractables can be obtained if the carbohydrate polymer is covalently attached to the paper fibers and/or is itself crosslinked. In some embodiments, the paper comprises 5-300 micromoles/g, such as 5-50, 5-100 or 50-300 micromoles/g of negatively or positively charged groups. Negatively charged groups can be, for example, carboxylate, sulfonate or sulfate groups, and positively charged groups can be, for example, amines or quaternary ammonium groups. The presence of these groups can improve the protective effect of the branched carbohydrate polymer.

[0099] The method for removing the sample can include storing the sample, eg, the dried paper bearing the biological sample, for at least one week, eg, at least one month or at least one year. In some embodiments, the method includes extracting at least one protein from the paper after storing and analyzing the proteins. Extraction can be done, for example, by punching out small pieces of paper with the dried sample and immersing them in an aqueous liquid. In some embodiments, the protein is analyzed in steps by immunoassay, mass spectroscopy or enzymatic activity assay. Examples of substrates disclosed above are disclosed, for example, in US Patent Application Publication No. 2014/0302521.

[0100] The sample stabilizing component can comprise at least one dried sample, eg, at least one dried biological sample, such as a dried blood sample. Applying blood and other biological materials such as serum, plasma, urine, cerebrospinal fluid, bone marrow, biopsies, etc. to a sample stabilizing component and drying for storage and subsequent analysis or other use. can be done. A dry sample, such as a biological sample, can be a pharmaceutical preparation or diagnostic reagent that contains at least one protein or other sensitive biomolecule. In another aspect, the sample stabilizing component can comprise a paper card with one or more sample application areas printed or otherwise indicated on the card. An indicator dye may be present in these areas to indicate whether an unpigmented sample has been applied. The device can include a card holder to facilitate automated handling, e.g., in a rack, and can include various forms of sampling features to facilitate sample collection. can.

[0101] Other examples of stabilizing matrices or stabilizing components that can be used in the devices herein include, but are not limited to, Gentegra-RNA further exemplified, Gentegra-DNA (Gentegra, Pleasanton Calif.), DNA Stable Plus as further exemplified in US Pat. No. 8,519,125; RNAgard Blood System (Biomatria, San Diego , CA).

[0102] In some embodiments, the solid matrix can selectively stabilize plasma constituents. Plasma constituents can include cell-free DNA, cell-free RNA, proteins, hormones, and other metabolites, which can be selectively stabilized on solid matrices. Plasma components can be isolated from whole blood and stabilized on a solid matrix. Solid matrices can overlap or be components of a variety of different devices and technologies. Plasma components can be separated from whole blood samples using a variety of different devices and techniques. Techniques may include lateral flow assays, vertical flow assays, and centrifugation.

[0103] The solid matrix can be integrated with or be a component of various plasma separation devices or technologies. The solid matrix can overlay or be a component of a variety of different devices such as plasma separation membranes, eg, Vivid™ plasma separation membranes. A solid matrix can partially overlap a plasma separation device, such as a plasma separation membrane. Examples of devices and techniques for plasma separation are U.S. Pat. Nos. 6,045,899; 5,906,742; 6,565,782; , 6,939,468, EP 0,946,354, 0,846,024, U.S. Patent 6,440,306, 6,110,369 Nos. 5,979,670, 5,846,422, 6,277,281, European Patent Specifications 1,118,377, 0,696,935, No. 1,089,077, U.S. Patent Specification Nos. 2013/0210078, 2015/0031035, and other patents or patent publications, which are incorporated herein by reference.

[0104] Separation membranes can be used in a variety of devices and techniques. Separation membranes can be constructed of polycarbonate, fiberglass, or others recognized by those skilled in the art. A membrane can include a solid matrix. The membrane can have variable pore sizes. The separation membrane can have pore diameters of about 1 μm, about 2 μm, about 4 μm, about 6 μm, about 8 μm, about 10 μm, about 12 μm, about 14 μm, about 16 μm, about 18 μm, about 20 μm. The separation membrane can have pores with diameters from about 2 μm to about 4 μm. The separation membrane can have pores with a diameter of about 2 μm.

[0105] Plasma separation can be performed on a wide variety of sample volumes. The plasma sample volume can be a variable depending on the application for which the solid matrix is used. The sample volume is about 100 μL, about 150 μL, about 200 μL, about 250 μL, about 300 μL, about 350 μL, about 400 μL, about 450 μL, about 500 μL, about 550 μL, about 600 μL, about 650 μL, about 700 μL, about 750 μL, about 800 μL, about It can be 850 μL, about 900 μL, about 950 μL, or about 1000 μL. Sample volumes can range from about 250 μL to about 500 μL.

[0106] Preparation of Biological Components

[0107] In some embodiments, after the user or operator has removed the components of the system for analysis, the sample can be sent to a facility for analysis, for example. The facility may be a CLIA facility, laboratory, clinic, or an external dedicated facility. At the institution, the samples may be used for any diagnostic test including, but not limited to, common panels, thyroid tests, cancer diagnostic tests, cardiovascular disease tests and screening, genetic/prenatal tests and infectious diseases. A non-limiting list of applicable tests is included herein as Figure 7 and co-filed.

[0108] Devices and systems for acquiring, collecting, separating and stabilizing samples may be modular and include separate compartments or components. Separate components can be easily removed or separated, or they cannot be removed or separated. A separable or removable component may include a sample substrate or a sample separation component.

[0109] The devices herein (eg, the sample acquisition component and the sample stabilization component) can be transported together, eg, to a laboratory for further analysis of the biological component. Alternatively, the stabilizing component (eg, substrate or substrate) can be shipped to the laboratory without the sample acquisition component for further analysis of the biological component. In some examples, treatments and analyzes can be performed on the deployed device, system or substrate, and either the stabilization component or the component of the device or system can be used by a healthcare provider or test results of interest. can be transported to others skilled in the art. Once the biological sample is received at the site for analysis, the substrate, or component of the sample separation component, can be removed. These components may be tagged and/or labeled to indicate the composition or target component stabilized on the matrix. Using tags as identifiers, membranes or substrates can be sorted and prepared for targeted testing.

[0110] Systems and processes for receiving and processing samples may vary depending on the identity, stability, source, or other characteristics of the sample placed. The quality of biological sample components can directly affect the quality, reproducibility and reliability of diagnostic test results. The aforementioned systems, devices and methods for sample acquisition, collection, stabilization and optional separation can affect sample composition, stability, concentration and processing. For example, the volume, size, quantity, stability and purity of a biological sample can vary depending on the sample source and the components used to collect the sample. The quality of the sample and also the quality of the diagnostic result can be kit, system, device and method specific for obtaining the sample. Accordingly, methods, kits and systems for receiving, processing, treating and/or preparing components of a biological sample prior to analysis are also disclosed.

[0111] The components processed from the sample include, but are not limited to, DNA/RNA such as cell-free DNA and circulating tumor DNA, proteins, antigens, antibodies, lipids such as HDL/LDL, and any of these. A combination of A biological sample or target component can be extracted using any conventional nucleic acid extraction method. Non-limiting examples of extraction methods include, but are not limited to, electroelution, gelatin extraction, silica or glass bead extraction, guanidine-thiocyanate-phenol solution extraction, guanidine thiocyanate extraction, sodium iodide or similar gradients. centrifugation, centrifugation with buffer, or phenol-chloroform based extraction. For nucleic acid analysis, an extraction step can help remove impurities such as proteins, concentrating circulating nucleic acids. Extracted circulating nucleic acids can be examined using methods such as agarose gel electrophoresis, spectrophotometry, fluorometry, or liquid chromatography.

[0112] DNA or nucleotides extracted from a sample can be prepared in the laboratory using a variety of methods to reduce error and generate significant signal with limited sample size. A nucleic acid sample can be treated or subjected to various methods for efficient amplification of a desired target nucleic acid (eg, a "DNA template" or "nucleic acid template"). Modified primers can be designed to minimize or prevent the formation of undesirable primer-dimer and chimeric products observed in other nucleic acid amplification methods and kits. Novel primer design methods can avoid the generation of spurious nucleic acid amplification products. The described methods and kits used to analyze samples can include "AT GenomiPhi." ATGenomiPhi can use modified hexamers of the general formula: +N+N(atN)(atN)(atN)*N, where the '+' precedes the LNA base, as described above, (atN) represents a random mixture of 2-amino-dA, dC, dG and 2-thio-dT. Other hexamers can have the formula (atN)(atN)(atN)(atN)(atN)*N, and the notation is consistent between these two hexamer designs. The use of these hexamers in nucleic acid amplification technology increases the melting _Tm of the primers by blocking the ability of random hexamers to anneal to each other, thereby increasing the ability of LNA and 2-amino-dA to primers. by improving the binding efficiency of the hexamer to the target nucleic acid through the addition of can address, minimize, or eliminate problems associated with primer-dimer formation and chimeric nucleic acid production observed in conventional methods. In addition, primer modifications can increase their binding strength to target nucleic acids, allowing the use of more stringent hybridization buffers that further minimize the potential for production of primer dimers and chimeric nucleic acid products. be possible. These and other methods of DNA amplification methods can be found, for example, in US Patent Application Publication No. 2013/0210078.

[0113] DNA from a sample can be analyzed using one or more methods for generating single-stranded DNA circles from a biological sample. A laboratory that analyzes a sample includes the steps of treating a biological sample with an extractant to release nucleic acids, thereby forming a sample mixture, neutralizing the extractant, and denaturing the released nucleic acids. contacting the single-stranded nucleic acid with a ligase capable of non-templated intramolecular ligation of single-stranded DNA sequences to produce single-stranded DNA circles; can be used. The method steps can be performed without any intermediate nucleic acid isolation or nucleic acid purification. In certain embodiments, steps may be performed in a sequential fashion within a single reaction vessel. In certain embodiments, single-stranded DNA circles can be amplified to allow subsequent analysis of biological samples. In certain embodiments, the sample mixture can be dried onto the solid matrix prior to the neutralization step. In certain embodiments, damage to DNA can be repaired enzymatically prior to the denaturation step. In another aspect, methods are provided for analyzing a sample, eg, a biological sample. Therefore, single-stranded DNA circles generated according to certain embodiments of this method are amplified and the amplification products are analyzed. Analysis can be performed, for example, by targeted sequencing of the amplification products. In another aspect, a method is provided for detecting breakpoints of chromosomal rearrangements from a biological sample. Thus, single-stranded DNA circles produced according to certain embodiments described herein are amplified and the amplification products analyzed, eg, by sequencing. Breakpoints of any chromosomal rearrangement can be identified by comparing the sequence to a known reference sequence. In yet another aspect, an extractant for treating a biological sample to release nucleic acids, a reagent for neutralizing the extractant, and allowing for template-independent intramolecular ligation of single-stranded DNA sequences. A kit can be provided that includes a ligase, and These and other methods of DNA amplification methods can be found, for example, in International Patent Application No. US2015/50760.

[0114] Methods for generating single-stranded DNA circles from linear DNA can be used on the collected sample. The method involves providing linear DNA and end-repairing the linear DNA by incubating it with polynucleotide kinase in the presence of a phosphate donor to form a phosphate group at the 5′ end and a phosphate group at the 3′ end. generating ligatable DNA sequences with terminal hydroxyl groups; and performing intramolecular ligation of the repaired ligatable DNA sequences with a ligase to generate single-stranded DNA circles. , can be included. These steps can be performed in a single reaction vessel without any intervening isolation or purification steps. The phosphate donor can be guanosine triphosphate (GTP), cytidine triphosphate (CTP), uridine triphosphate (UTP), deoxythymidine triphosphate (dTTP) or combinations thereof. Linear DNA can be either double-stranded or single-stranded DNA. The DNA can be compartments of fragmented DNA, such as circulating DNA. The ligatable DNA, if in double-stranded form, may need to be denatured prior to the intramolecular ligation reaction. The ligation reaction can employ a pre-adenylation ligase capable of template-independent intramolecular ligation of single-stranded DNA sequences. In other embodiments, methods for generating single-stranded DNA circles from linear DNA can employ a DNA pre-adenylation step prior to the intramolecular ligation step. The linear DNA is optionally incubated with polynucleotide kinase in the presence of adenosine triphosphate (ATP) to produce ligatable DNA containing a phosphate group at the 5' end and a hydroxyl group at the 3' end. Arrays can be generated. If the linear DNA is in a highly fragmented form, it is preferable to generate ligatable DNA sequences from the linear DNA. Linear DNA or ligatable DNA sequences can then be incubated with an adenylating enzyme in the presence of ATP to generate 5' adenylated DNA sequences. The 5'-adenylated DNA sequences can be incubated with a non-adenylating ligase, which is capable of template-independent intramolecular ligation of the 5'-adenylated DNA sequences to generate single-stranded DNA circles. can do. All steps of the method can be performed in a single reaction vessel without any intervening isolation or purification steps. If the non-adenylating ligase is an ATP-dependent ligase, ATP can be removed from the reaction mixture (eg, by treating the reaction mixture with a phosphatase) prior to the intramolecular ligation reaction. If the 5'-adenylated DNA is in double-stranded form, it may need to be denatured prior to the intramolecular ligation reaction. These and other methods of DNA circularization and amplification methods can be found, for example, in US Patent Application Publication No. 2015/0031086.

[0115] Sample processing can include preparing RNA for analysis. The method may include use in the production of nucleic acid structures and subsequent purification and amplification of nucleic acids. This method may require a DNA sequence containing double-stranded and single-stranded regions. The single-stranded region can be complementary to the RNA sequence of interest. An RNA sequence can then be hybridized to the single-stranded region of the DNA sequence and the two sequences then ligated to produce an RNA-DNA molecule. The method can include a step whereby the 3' end of the RNA is ligated to a double-stranded DNA oligonucleotide containing a promoter sequence. This double-stranded DNA oligonucleotide can contain a promoter for RNA polymerase within the double-stranded region, followed by a segment of single-stranded DNA that forms a 3' overhang. A single-stranded portion of double-stranded DNA can hybridize to the 3' end of a messenger RNA (mRNA) poly(A) tail if the 3' overhang contains a string of thymidine residues. After addition of ligase, the mRNA may have one strand of double-stranded DNA sequence ligated to the 3' end. Addition of RNA polymerase allows efficient transcription of RNA-DNA hybrid molecules to synthesize cRNA. This method allows efficient RNA amplification because the transcription reaction using RNA polymerase can transcribe each template multiple times. Another method similar to that described above may involve ligating a DNA oligonucleotide to RNA as described above. However, DNA oligonucleotides can either be attached to a solid support or contain an affinity tag. This allows for very efficient covalent binding and/or trapping of RNA molecules and can be used for any number of different purposes. Further methods can utilize ligation and subsequent transcription to create complementary RNAs containing user-defined sequences at the 5' end of the cRNA. This sequence "tag" can be placed between the RNA polymerase promoter and the 3' end of the ligated RNA molecule. User-defined sequences can be used for cRNA purification or identification or other sequence-specific manipulations. Subsequently, when the cRNA product is subsequently ligated and re-amplified according to the described method, the resulting double-stranded amplification product may be sense with respect to the original sense template, and this new product may have two distinct user-defined sequences located at the 5' ends. These sequences can be used for cDNA synthesis, allowing full-length synthesis and directional cloning. Those skilled in the art will appreciate that this double amplification method, with or without user-defined sequences, can result in significant increases in RNA abundance, allowing analysis of samples that were previously too small to consider. You will understand that it is possible. These and other methods for amplification of DNA fragments can be found, for example, in US Patent Application Publication No. 2008/0003602.

[0116] A further method for analyzing a biological sample focuses on nucleic acids present in regions of interest in the biological sample, provides protein expression information for many proteins, and augments DNA sequence information. can do. Additional methods for sample analysis can focus on homogeneous subsections of heterogeneous samples. Particular subpopulations of cells within a mixed population can be precisely identified and analyzed from predetermined selection criteria. Mutations can be identified that may be useful for diagnostic and/or prognostic or further investigation of drug targets. These and other methods for DNA amplification are found, for example, in International Application No. US2015/50760.
Nucleic acid elution

[0117] In some cases, nucleic acids, such as DNA or RNA, in a sample (e.g., biological sample) are applied to a stabilizing matrix (e.g., a nucleic acid stabilizing matrix), and the sample is optionally dried onto the stabilizing matrix. and the nucleic acids, such as DNA or RNA, on the stabilizing matrix are eluted from the stabilizing matrix. In some examples, the dried biological sample is stabilized on a stabilizing matrix capable of stabilizing nucleic acids, eg, as described herein. In some examples, the method comprises (a) contacting, e.g., spotting, a sample, e.g., biological sample, comprising nucleic acids, e.g., DNA or RNA, on a nucleic acid-stabilized matrix; (c) optionally contacting the nucleic acid stabilizing matrix comprising the nucleic acid with a lysis buffer; (d) optionally, e.g. and (e) optionally, (f) contacting the nucleic acid stabilizing matrix comprising the nucleic acid with a wash buffer, for example by soaking the nucleic acid stabilizing matrix in the wash buffer; contacting a nucleic acid stabilizing matrix containing nucleic acids with an elution buffer by soaking to elute the nucleic acids from the stabilizing matrix.

[0118] In some cases, nucleic acids, such as RNA or DNA, can be extracted from a stabilizing matrix, such as a paper stabilizing matrix, using, for example, an extraction buffer. Extracted nucleic acids, such as RNA or DNA, can be bound to a second matrix, such as a second solid support, such as beads, such as magnetic beads. Binding to a second matrix, eg, a second solid support, eg, beads, eg, magnetic beads, can occur in a binding buffer. Nucleic acids, such as RNA or DNA, on the second matrix can be washed with one or more wash buffers. Nucleic acids on the second matrix can be treated with enzyme solutions containing, for example, DNase, RNase, or proteases to degrade specific types of molecules (eg, RNA, DNA, or proteins). Nucleic acids on a solid matrix can be eluted from the solid matrix using, for example, an elution buffer.

[0119] Elution can be performed on at least a portion of a stabilizing matrix that includes a sample (eg, a dried biological sample). In some cases, a portion of the stabilization matrix can be separated from the remainder of the stabilization matrix, e.g., a portion of the stabilization matrix can be punched out of the stabilization matrix and the nucleic acids in the separated portion can be eluted. can. The punched portion is approximately 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 mm in diameter may be The stamped portion has a diameter of about 10 to about 60, about 10 to about 50, about 10 to about 40, about 10 to about 30, about 10 to about 20, about 1 to about 10, about 2 to about 9, about 3 to about 8, about 4 to about 7, about 5 to about 6, about 3 to about 6, about 1 to about 4, about 1 to about 3, or about 1 to about 2 mm. Optionally, the nucleic acid is eluted from the stabilizing matrix after the stabilizing matrix containing the nucleic acid has been separated into portions.

[0120] Optionally, a nucleic acid-stabilized matrix comprising nucleic acids can be contacted with a lysis buffer. In some cases, nucleic acid binding and retention to the stabilizing matrix can be enhanced by contacting the stabilizing matrix with a binding buffer. Optionally, a portion of the stabilizing matrix is contacted with a nucleic acid binding buffer. Optionally, the nucleic acid binding buffer can contain beads. In some cases, the stabilizing matrix is contacted with a wash buffer, eg, to remove impurities. Optionally, nucleic acids can be eluted by contacting the stabilizing matrix with an elution buffer.

[0121] In some cases, the nucleic acid lysis buffer, binding buffer, wash buffer, or elution buffer can comprise commercially available buffers. For example, buffers include TRIzol® (Thermofisher®), Buffer RLT (Qiagen®), Buffer RLN (Qiagen®), RNA Lysis Buffer (RLA) (Promega PureYield™ Cell Lysis Solution (CLA) (Promega), PureYield™ Endotoxin Removal Wash Solution (Promega), PureZOL™ RNA Isolation Reagent (Bio-Rad™), RNA Lysis Buffer or DNA/RNA Binding Buffer (Zymo Research Corp), or RNA Acquisition Buffer (Pierce™) may be included.

[0122] Optionally, the nucleic acid lysis buffer, binding buffer, wash buffer, or elution buffer contains one or more buffers (or pH buffers), one or more salts, one or more reducing buffers. one or more chelating agents, one or more surfactants, one or more enzymes, one or more protein denaturants, one or more blocking reagents, one or more organic solvents, or any of these It may also include combinations. For example, a nucleic acid lysis buffer, binding buffer, wash buffer, or elution buffer can contain one or more protein denaturants and one or more reducing agents. Nucleic acid lysis buffers, binding buffers, wash buffers, or elution buffers can include one or more protein denaturants, one or more reducing agents, and one or more enzymes. A nucleic acid lysis buffer, binding buffer, wash buffer, or elution buffer comprising one or more buffers, one or more protein denaturants, one or more reducing agents, and one or more enzymes can be done. Nucleic acid lysis buffers, binding buffers, wash buffers, or elution buffers can include one or more salts and one or more buffers. Nucleic acid lysis buffers, binding buffers, wash buffers, or elution buffers can include one or more salts, one or more buffers and one or more organic solvents. Nucleic acid lysis buffers, binding buffers, wash buffers, or elution buffers can include one or more buffers and one or more enzymes. Nucleic acid lysis buffers, binding buffers, wash buffers, or elution buffers can include one or more buffers and one or more chelating agents. Nucleic acid lysis buffers, binding buffers, wash buffers, or elution buffers can include one or more buffers, one or more chelating agents and one or more organic solvents.

[0123] The one or more buffering agents are, for example, saline, citrate, phosphate, phosphate buffered saline, acetate, glycine, tris(hydroxymethyl)aminomethane (tris) hydrochloride, tris buffered saline (TBS), 3-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]propane-1-sulfonic acid (TAPS), bicine, tricine, 3-[[ 1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]-2-hydroxypropane-1-sulfonic acid (TAPSO), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid ( HEPES), piperazine-N, N′-bis(2-ethanesulfonic acid) (PIPES), 3-(N-morpholino)propanesulfonic acid (MOPS), 2-(N-morpholino)ethanesulfonic acid (MES), It can be 2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid (TES), cacodylate, glycine, carbonate, or any combination thereof. The one or more buffering agents is about 0.1 mM to about 500 mM, about 0.1 mM to about 400 mM, about 0.1 mM to about 300 mM, about 0.1 mM to about 200 mM, about 0.1 mM to about 100 mM, about 0 .1 mM to about 50 mM, about 0.1 mM to about 25 mM, about 0.1 mM to about 20 mM, about 0.1 mM to about 15 mM, about 0.1 mM to about 10 mM, about 0.1 mM to about 5 mM, about 0.1 mM to about 4 mM, about 0.1 mM to about 3 mM, about 0.1 mM to about 2 mM, about 0.1 mM to about 1 mM, about 0.1 mM to about 0.9 mM, about 0.1 mM to about 0.8 mM, about 0 .1 mM to about 0.7 mM, about 0.1 mM to about 0.6 mM, about 0.1 mM to about 0.5 mM, about 0.1 mM to about 0.4 mM, about 0.1 mM to about 0.3 mM, or about It may be present at a concentration of 0.1 mM to about 0.2 mM. Buffer is less than 500 mM, less than 400 mM, less than 300 mM, less than 200 mM, less than 100 mM, less than 50 mM, less than 25 mM, less than 20 mM, less than 15 mM, less than 10 mM, less than 5 mM, less than 4 mM, less than 3 mM, less than 2 mM, less than 1 mM, 0 present at a concentration of less than .9 mM, less than 0.8 mM, less than 0.7 mM, less than 0.6 mM, less than 0.5 mM, less than 0.4 mM, less than 0.3 mM, less than 0.2 mM, or less than 0.1 mM good too. Buffering agents greater than 500 mM, greater than 400 mM, greater than 300 mM, greater than 200 mM, greater than 100 mM, greater than 50 mM, greater than 25 mM, greater than 20 mM, greater than 15 mM, greater than 10 mM, greater than 5 mM, greater than 4 mM, greater than 3 mM, greater than 2 mM, greater than 1 mM, 0 may be present at concentrations greater than .9 mM, greater than 0.8 mM, greater than 0.7 mM, greater than 0.6 mM, greater than 0.5 mM, greater than 0.4 mM, greater than 0.3 mM, greater than 0.2 mM, or greater than 0.1 mM .

[0124] The one or more salts are, for example, sodium chloride, sodium acetate, sodium bicarbonate, sodium bisulfate, sodium bromide, potassium chloride, potassium acetate, potassium bicarbonate, potassium bisulfate, potassium bromate, potassium bromide Potassium, or potassium carbonate. The one or more salts can be at a concentration of about 0.1 mM, 5 mM, 10 mM, 25 mM, 50 mM, 100 mM, 250 mM, 500 mM, 750 mM or 1000 mM in the buffer. One or more salts can be at a concentration of less than 0.1 mM, 5 mM, 10 mM, 25 mM, 50 mM, 100 mM, 250 mM, 500 mM, 750 mM or 1000 mM in the buffer. One or more salts can be at a concentration of at least 0.1 mM, 5 mM, 10 mM, 25 mM, 50 mM, 100 mM, 250 mM, 500 mM, 750 mM or 1000 mM.

[0125] The one or more reducing agents are, for example, β-mercaptoethanol (BME), 2-aminoethanethiol (2MEA-HCl (cysteamine-HCl)), dithiothreitol (DTT), glutathione (GSH), tris (2-carboxyethyl)phosphine (TECP), or any combination thereof. The concentration of one or more reducing agents can be about 0.1 mM, 0.5 mM, 1 mM, 10 mM, 50 mM, 100 mM, 250 mM or 500 mM. The concentration of one or more reducing agents can be less than 0.5 mM, 1 mM, 10 mM, 50 mM, 100 mM, 250 mM, or 500 mM. For example, the concentration of DTT can be about 0.05 mM to about 100 mM, about 0.5 mM to about 50 mM, or about 5 mM to about 10 mM. The concentration of TCEP can be about 0.05 mM to about 50 mM, about 0.5 mM to about 50 mM, or about 0.5 mM to about 5 mM. The concentration of BME can be from about 0.05% to about 10%, from about 0.5% to about 5%, or from about 1% to about 10%. The concentration of GSH can be about 0.05 mM to about 25 mM, about 0.5 mM to about 10 mM, or about 5 mM to about 10 mM. The concentration of the one or more reducing agents is about 1 mM, 10 mM, 50 mM, 100 mM, 250 mM, or 500 mM.

[0126] The one or more chelating agents are, for example, carbohydrates, lipids, steroids, amino acids or related compounds, phosphates, nucleotides, tetrapyrroles, ferrioxamines, ionophores, phenolic chelating agents, or synthetic chelating agents, such as 2,2'-bipyridyl, dimercaptopropanol, ethylenediaminetetraacetic acid (EDTA), ethylenedioxy-diethylene-dinitrilo-tetraacetic acid, ethylene glycol-bis-(2-aminoethyl)-N,N,N',N' - can be tetraacetic acid (EGTA), metal nitrilotriacetic acid (NTA), salicylic acid, or triethanolamine (TEA). The concentration of one or more chelating agents in the buffer can be about 0.1 mM, 1 mM, 5 mM, 10 mM, 20 mM or 25 mM. The concentration of one or more chelating agents in the buffer can be less than 0.1 mM, 1 mM, 5 mM, 10 mM, 20 mM or 25 mM. The concentration of one or more chelating agents in the buffer can be greater than 0.1 mM, 1 mM, 5 mM, 10 mM, 20 mM or 25 mM.

[0127] The one or more surfactants can be, for example, anionic, cationic, nonionic, or amphoteric. The one or more surfactants are polyethoxylated alcohols, polyoxyethylene sorbitan, octoxynol such as Triton X100™ (polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether) , polysorbates such as Tween™ 20 (e.g., polysorbate 20) or Tween™ 80 (polysorbate 80), sodium dodecyl sulfate, sodium lauryl sulfate, nonylphenol ethoxylates such as Tergitol™, cyclodextrins, or these It can be any combination. One or more surfactants is less than 0.001 vol.%, less than 0.005 vol.%, less than 0.01 vol.%, less than 0.015 vol.%, 0.02 vol.% of the total volume of the elution buffer. Less than 0.025% by volume, less than 0.03% by volume, less than 0.035% by volume, less than 0.04% by volume, less than 0.045% by volume, less than 0.05% by volume, 0.055% by volume %, less than 0.06% by volume, less than 0.065% by volume, less than 0.07% by volume, less than 0.075% by volume, less than 0.08% by volume, less than 0.085% by volume, 0.09% by volume Less than, less than 0.095% by volume, less than 0.1% by volume, less than 0.15% by volume, less than 0.2% by volume, less than 0.25% by volume, less than 0.3% by volume, less than 0.35% by volume , less than 0.4% by volume, less than 0.45% by volume, less than 0.5% by volume, less than 0.55% by volume, less than 0.6% by volume, less than 0.65% by volume, less than 0.7% by volume; It may be present at a concentration of less than 0.75%, less than 0.8%, less than 0.85%, less than 0.9%, or less than 0.1% by volume. The one or more surfactants can be at concentrations of about 0.01%, 0.05%, 0.1%, 0.5%, 1%, 5% or 10%. The one or more surfactants can be at concentrations less than 0.01%, 0.05%, 0.1%, 0.5%, 1%, 5% or 10%. The one or more surfactants can be at concentrations greater than 0.01%, 0.05%, 0.1%, 0.5%, 1%, 5% or 10%.

[0128] The one or more protein denaturing agents can be, for example, a chaotropic agent, such as a chaotropic salt. Chaotropic agents include butanol, ethanol, guanidine chloride, guanidine hydrochloride, guanidine isothiocyanate, lithium perchlorate, lithium acetate, magnesium chloride, phenol, propanol, sodium iodide, sodium thiocyanate, thiourea, urea or any of them. It can be a combination. The concentration of chaotropic agent in the buffer can be about 0.1 mM, 1 mM, 10 mM, 100 mM, 1 M, 6 M or 8 M. The concentration of chaotropic agent in the buffer can be at least 0.1 mM, 1 mM, 10 mM, 100 mM, 1 M, 6 M or 8 M. The concentration of chaotropic agent in the buffer can be less than 0.1 mM, 1 mM, 10 mM, 100 mM, 1 M, 6 M or 8 M.

[0129] Nucleic acid lysis buffers, binding buffers, wash buffers or elution buffers can further comprise one or more enzymes. Lysis buffers, binding buffers, wash buffers or elution buffers can contain sufficient amounts of DNase or RNase to remove DNA or RNA impurities, respectively, from each other. Lysis buffers, binding buffers, wash buffers or elution buffers may contain lytic enzymes such as chicken egg white lysozyme, T4 lysozyme, as well as carbohydrase, phytase and trypsin, proteinase K, pepsin, chymotrypsin, papain, bromelain, subtilisin, or elastase. may include enzymes such as proteases such as The protease can be a serine protease, a cysteine protease, a threonine protease, an aspartic protease, a glutamic protease, or a metalloprotease, or an asparagine peptide lyase.

[0130] Nucleic acid lysis buffers, binding buffers, wash buffers or elution buffers are about 2 to about 10, about 2 to about 9, about 2 to about 8, about 2 to about 7, about 2 to about 6 , about 2 to about 5, about 2 to about 4, or about 2 to about 3. The nucleic acid lysis buffer, binding buffer, wash buffer or elution buffer is about 6 to about 8, about 6 to about 7.9, about 6 to about 7.8, about 6 to about 7.7, about 6 to about 7.6, about 6 to about 7.5, about 6 to about 7.4, about 6 to about 7.3, about 6 to about 7.2, about 6 to about 7.1, about 6 to about 7, about 6 to about 6.9, about 6 to about 6.8, about 6 to about 6.7, about 6 to about 6.6, about 6 to about 6.5, about 6 to about 6.4, It can be an aqueous solution having a pH of about 6 to about 6.3, about 6 to about 6.2, or about 6 to about 6.1. The nucleic acid lysis buffer, binding buffer, wash buffer or elution buffer comprises at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 5, at least about 8, at least about 9, Or it can be an aqueous solution having a pH of at least about 10.

[0131] Nucleic acid lysis buffers, binding buffers, wash buffers, or elution buffers can further comprise additional components. For example, lysis buffers, binding buffers, wash buffers, or elution buffers can contain protein blocking agents to minimize non-specific binding, such as bovine serum albumin, fetal bovine serum, and the like. Lysis buffers, binding buffers, wash buffers, or elution buffers may contain bacterial DNA, bacterial DNA, yeast DNA, yeast RNA, mammalian nucleic acids, including primate nucleic acids such as human or chimpanzee DNA, or herring, mackerel. Additional nucleic acid (including DNA or RNA) from a different organism than the subject can be included, such as non-mammalian nucleic acid, including nucleic acid from fish such as fish, krill, or salmon DNA.

[0132] Lysis buffers, binding buffers, wash buffers, or elution buffers can include an amount of one or more organic solvents, eg, to enhance binding of nucleic acids to the stabilizing matrix. The one or more organic solvents can be methanol, ethanol, DMSO, DMF, dioxane, tetrahydrofuran, propanol, isopropanol, butanol, t-butanol or pentanol, acetone, and the like. Optionally, the binding buffer is less than 0.01 vol.%, less than 0.05 vol.%, less than 0.1 vol.%, less than 0.15 vol.%, 0.2 vol.% of the total volume of the solution. Less than, less than 0.25% by volume, less than 0.3% by volume, less than 0.35% by volume, less than 0.4% by volume, less than 0.45% by volume, less than 0.5% by volume, less than 0.55% by volume , less than 0.6% by volume, less than 0.65% by volume, less than 0.7% by volume, less than 0.75% by volume, less than 0.8% by volume, less than 0.85% by volume, less than 0.9% by volume; 4. less than 0.95% by volume, less than 1% by volume, less than 1.5% by volume, less than 2% by volume, less than 2.5% by volume, less than 3% by volume, less than 3.5% by volume, less than 4% by volume; Less than 5% by volume, less than 5% by volume, less than 5.5% by volume, less than 6% by volume, less than 6.5% by volume, less than 7% by volume, less than 7.5% by volume, less than 8% by volume, 8.5% by volume %, less than 9%, less than 9.5%, less than 10%, less than 11%, less than 12%, less than 13%, less than 14%, less than 15%, less than 16% by volume, less than 17% by volume, less than 18% by volume, less than 19% by volume, less than 20% by volume, less than 25% by volume, less than 30% by volume, less than 35% by volume, less than 40% by volume, less than 45% by volume, less than 50% by volume; less than 55% by volume, less than 60% by volume, less than 65% by volume, less than 70% by volume, less than 75% by volume, less than 80% by volume, less than 85% by volume, less than 90% by volume, less than 95% by volume, less than 99% by volume; or may contain less than 100% by volume of organic solvent. The organic solvent can be at concentrations of at least 0.1%, 1%, 10%, 50%, 75% or 100%. Organic solvents can be at concentrations of about 0.1%, 1%, 10%, 50%, 75% or 100%.

[0133] The stabilization matrix or a portion of the stabilization matrix is less than 5 μL, less than 10 μL, less than 15 μL, less than 20 μL, less than 25 μL, less than 30 μL, less than 35 μL, less than 40 μL, less than 45 μL, less than 50 μL, less than 55 μL, less than 60 μL, <65 μL, <70 μL, <75 μL, <80 μL, <85 μL, <90 μL, <95 μL, <100 μL, <110 μL, <120 μL, <130 μL, <140 μL, <150 μL, <160 μL, <170 μL, <180 μL, <190 μL , <200 μL, <250 μL, <300 μL, <350 μL, <400 μL, <450 μL, <500 μL, <550 μL, <600 μL, <650 μL, <700 μL, <750 μL, <800 μL, <850 μL, <900 μL, <950 μL, 1 < ,000 μL, <1.5 mL, <2 mL, <2.5 mL, <3 mL, <3.5 mL, <4 mL, <4.5 mL, <5 mL, <5.5 mL, <6 mL, <6.5 mL, 7 mL Less than, less than 7.5 mL, less than 8 mL, less than 8.5 mL, less than 9 mL, less than 9.5 mL, or less than 10 mL volume of nucleic acid binding buffer, wash buffer or elution buffer can be contacted. The stabilization matrix or portion of the stabilization matrix can be contacted with about 0.1 mL, 0.2 mL, 0.5 mL, 0.7 mL, 1 mL, 2 mL, 5 mL, 7 mL, or 10 mL of buffer. The stabilizing matrix or a portion of the stabilizing matrix can be contacted with at least 0.1 mL, 0.2 mL, 0.5 mL, 0.7 mL, 1 mL, 2 mL, 5 mL, 7 mL or 10 mL of buffer.

[0134] The volume of binding buffer, wash buffer, or elution buffer that contacts the stabilizing matrix can depend on the surface area of the stabilizing matrix. The amount of binding buffer, wash buffer, or elution buffer is less than 1 μL/ ^mm2 , less than ² μL/ ^mm2 , less than 3 μL/ ^mm2 , less than 4 μL/ ^mm2 , less than 5 μL/mm2, 6 μL/ ^mm2 less than 7 μL/mm ² , less than 8 μL/mm ² , less than 9 μL/mm ² , less than 10 μL/mm ² , less than 12 μL/mm ² , less than 14 μL/mm ² , less than 16 μL/mm ² , less than 18 μL/mm ² , less than 20 μL/ ^mm2 , less than 25 μL/ ^mm2 , less than 30 μL/ ^mm2 , less than 35 μL/ ^mm2 , less than 40 μL/ ^mm2 , less than 45 μL/ ^mm2 , less than 50 μL/ ^mm2 , less than 55 μL/ ^mm2 , 60 μL/mm2 ^mm2 , less than 65 μL/ ^mm2 , less than 70 μL/ ^mm2 , less than 75 μL/ ^mm2 , less than 80 μL/ ^mm2 , less than 85 μL/ ^mm2 , less than 90 μL/ ^mm2 , less than 95 μL/ ^mm2 , 100 μL/ ^mm2 less than, less than 150 μL/ ^mm2 , less than 200 μL/ ^mm2 , less than 250 μL/ ^mm2 , less than 300 μL/ ^mm2 , less than 350 μL/ ^mm2 , less than 400 μL/ ^mm2 , less than 450 μL/ ^mm2 , less than 500 μL/ ^mm2 , less than 550 μL/ ^mm2 , less than 600 μL/ ^mm2 , less than 650 μL/ ^mm2 , less than 700 μL/ ^mm2 , less than 750 μL/ ^mm2 , less than 800 μL/ ^mm2 , less than 850 μL/ ^mm2 , less than 900 μL/ ^mm2 , 950 μL/mm2 mm ² or less than 1,000 μL/mm ² . In some cases, the amount of binding buffer, wash buffer, or elution buffer is about 10 μL/mm ² to about 1,000 μL/mm ² , about 10 μL/mm ² to about 900 μL/mm ² , about 10 μL/mm ² to about 800 μL/mm ² , about 10 μL/mm ² to about 700 μL/mm ² , about 10 μL/mm ² to about 600 μL/mm ² , about 10 μL/mm ² to about 500 μL/mm ² , about 10 μL/mm ² to about 400 μL/mm ² , about 10 μL/mm ² to about 300 μL/mm ² , about 10 μL/mm ² to about 200 μL/mm ² , about 10 μL/mm ² to about 100 μL/mm ² , about 10 μL/mm ² to about 90 μL /mm ² , about 10 μL/mm ² to about 80 μL/mm ² , about 10 μL/mm ² to about 70 μL/mm ² , about 10 μL/mm ² to about 60 μL/mm ² , about 10 μL/mm ² to about 50 μL/mm ² , from about 10 μL/mm ² to about 40 μL/mm ² , from about 10 μL/mm ² to about 30 μL/mm ² , or from about 10 μL/mm ² to about 20 μL/mm ² .

[0135] Lysing, binding, washing or eluting nucleic acids can be performed in the presence or absence of agitation from a source of agitation. Agitation sources can be rockers, vortex mixers, mixers, shakers, and the like. In some cases, the agitation source can be set at a constant speed. The speed is less than 1 revolution per minute (rpm), less than 5 rpm, less than 10 rpm, less than 15 rpm, less than 20 rpm, less than 25 rpm, less than 30 rpm, less than 35 rpm, less than 40 rpm, less than 45 rpm, less than 50 rpm, less than 55 rpm, less than 60 rpm, less than 65 rpm , less than 70 rpm, less than 75 rpm, less than 80 rpm, less than 85 rpm, less than 90 rpm, less than 95 rpm, less than 100 rpm, less than 150 rpm, less than 200 rpm, less than 250 rpm, less than 300 rpm, less than 350 rpm, less than 400 rpm, less than 450 rpm, less than 500 rpm, less than 550 rpm, 600 rpm less than, less than 650 rpm, less than 700 rpm, less than 750 rpm, less than 800 rpm, less than 850 rpm, less than 900 rpm, less than 950 rpm, less than 1,000 rpm, less than 1,500 rpm, less than 2,000 rpm, less than 2,500 rpm, less than 3,000 rpm, 3, less than 500 rpm, less than 4,000 rpm, less than 4,500 rpm, less than 5,000 rpm, less than 5,500 rpm, less than 6,000 rpm, less than 6,500 rpm, less than 7,000 rpm, less than 7,500 rpm, less than 8,000 rpm; It can be less than 500 rpm, less than 9,000 rpm, less than 9,500 rpm, or less than 10,000 rpm. The speed can be about 50 rpm, 100 rpm, 200 rpm, 300 rpm, 400 rpm, 500 rpm, 600 rpm, 700 rpm, 800 rpm, 900 rpm, 1000 rpm, 1500 rpm or 5000 rpm. The speed can be at least 50 rpm, 100 rpm, 200 rpm, 300 rpm, 400 rpm, 500 rpm, 600 rpm, 700 rpm, 800 rpm, 900 rpm, 1000 rpm, 1500 rpm or 5000 rpm.

[0136] Binding, washing or eluting is performed at about less than 0°C, less than 1°C, less than 2°C, less than 3°C, less than 4°C, less than 5°C, less than 6°C, less than 7°C. , less than 8°C, less than 9°C, less than 10°C, less than 11°C, less than 12°C, less than 13°C, less than 14°C, less than 15°C, less than 16°C, less than 17°C , below 18°C, below 19°C, below 20°C, below 21°C, below 22°C, below 23°C, below 24°C, below 25°C, below 26°C, below 27°C , less than 28°C, less than 29°C, less than 30°C, less than 31°C, less than 32°C, less than 33°C, less than 34°C, less than 35°C, less than 36°C, less than 37°C , below 38°C, below 39°C, below 40°C, below 45°C, below 50°C, below 55°C, below 60°C, below 65°C, below 70°C, below 75°C , less than 80°C, less than 85°C, less than 90°C, less than 95°C, or about 100°C. Optionally, the binding, washing or elution is performed at about 10°C to about 100°C, about 10°C to about 95°C, about 10°C to about 90°C, about 10°C to about 85°C. , about 10°C to about 80°C, about 10°C to about 75°C, about 10°C to about 70°C, about 10°C to about 65°C, about 10°C to about 60°C , about 10°C to about 55°C, or about 10°C to about 50°C. Optionally, the binding, washing or elution is performed at about 20°C to about 50°C, about 20°C to about 48°C, about 20°C to about 46°C, about 20°C to about 44°C. , about 20°C to about 42°C, about 20°C to about 40°C, about 20°C to about 38°C, about 20°C to about 36°C, about 20°C to about 34°C , about 20°C to about 32°C, about 20°C to about 30°C, about 20°C to about 28°C, about 20°C to about 26°C, about 20°C to about 24°C , or at a temperature of about 20°C to about 22°C. The temperature can be about 10°C, 20°C, 25°C, 30°C, 37°C, 50°C, or 65°C.

[0137] Lyse, bind, wash or elute for less than 1 minute, less than 5 minutes, less than 10 minutes, less than 15 minutes, less than 20 minutes, less than 25 minutes, less than 30 minutes, less than 35 minutes, less than 40 minutes, 45 minutes It can be performed for less than, less than 50 minutes, less than 55 minutes, or less than 60 minutes. Binding, Washing or Elution is less than 0.5 hours, less than 1 hour, less than 1.5 hours, less than 2 hours, less than 2.5 hours, less than 3 hours, less than 3.5 hours, less than 4 hours, 4.5 hours hours, less than 5 hours, less than 5.5 hours, less than 6 hours, less than 6.5 hours, less than 7 hours, less than 7.5 hours, less than 8 hours, less than 8.5 hours, less than 9 hours, 9.5 less than 10 hours, less than 10.5 hours, less than 11 hours, less than 11.5 hours, or less than 12 hours, less than 18 hours, less than 1 day, less than 1.5 days, less than 2 days, 2.5 days Less than, less than 3 days, less than 3.5 days, less than 4 days, less than 4.5 days, less than 5 days, less than 5.5 days, less than 6 days, less than 6.5 days, or less than 7 days can be done. Lyse, bind, wash, or elute for about 0.25 hours, 0.5 hours, 1 hour, 2 hours, 5 hours, 10 hours, 12 hours, 18 hours, 24 hours, 3 days or 1 week, or more. Can run longer. Lysis, binding, washing or elution should be performed for at least 0.25 hours, 0.5 hours, 1 hour, 2 hours, 5 hours, 10 hours, 12 hours, 18 hours, 24 hours, 3 days, or 1 week can be done.

[0138] The eluted nucleic acid can be transferred to a container for storage or further processing, or can be transferred to an assay container for characterization.
Protein elution

[0139] Proteins can be eluted from matrices, such as the matrices described herein. A sample, eg, a biological sample, can be stabilized, prior to elution, on a stabilizing matrix that can stabilize proteins, eg, as described herein. Elution can include contacting the stabilizing matrix with an elution buffer. Contacting can include incubating (eg, with agitation) the stabilizing matrix in an elution buffer to elute the protein from the stabilizing matrix. Prior to elution, the stabilizing matrix can be contacted with a binding buffer or a washing buffer.

[0140] Elution can be performed on at least a portion of a stabilizing matrix that includes a sample (eg, a dried biological sample). Optionally, a portion of the stabilization matrix can be separated from the rest of the stabilization matrix and used for further processing. In some cases, the portion is greater than 5%, 15%, 25%, 35%, 45%, 55%, 65%, 75%, 85%, or 95% of the stabilizing matrix. Optionally, the portion is less than 5%, 15%, 25%, 35%, 45%, 55%, 65%, 75%, 85%, or 95% of the stabilizing matrix. A portion of the stabilizing matrix can be punched out of the stabilizing matrix and the protein in the separated portion can be eluted. The portion separated for further processing may comprise 100%, or about 90%, 80%, 70%, 60%, 50% or less of the sample applied to the matrix. The punched portion is approximately 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 mm in diameter can be The stamped portions have diameters up to about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 mm. The punched portion has a diameter of about 10 to about 60, about 10 to about 60, about 10 to about 60, about 10 to about 30, about 10 to about 20, about 1 to about 10, about 2 to about 9, about 3 to It can be about 8, about 4 to about 7, about 3 to about 6, about 4 to about 5, about 1 to about 4, about 1 to about 3, or about 1 to about 2 mm. In some cases, nucleic acids are eluted from the stabilization matrix after separation of the protein-containing stabilization matrix into portions.

[0141] The protein can be eluted from the stabilization matrix or portion of the stabilization matrix by contacting the stabilization matrix or portion of the stabilization matrix with a suitable elution buffer. The elution buffer comprises one or more buffering agents, one or more surfactants, one or more polyols, one or more salts, one or more blocking agents, one or more reducing agents, one or more Including an organic solvent, one or more chelating agents, such as one or more salts described herein, or any combination thereof. For example, an elution buffer can include one or more buffers and one or more polyols. Elution buffers can include one or more buffers and one or more surfactants. Elution buffers can include one or more buffers and one or more salts. Elution buffers can include one or more buffers and one or more reducing agents. Elution buffers can include one or more buffers, one or more surfactants, and one or more polyols. Elution buffers can include one or more buffers, one or more surfactants, and one or more salts. Elution buffers can include one or more buffers, one or more salts, and one or more reducing agents. Elution buffers can include one or more buffers, one or more surfactants, and one or more chelating agents. Elution buffers can include one or more buffers, one or more salts, one or more reducing agents, one or more chelating agents, one or more surfactants, and one or more polyols. can.

[0142] The one or more buffering agents are, for example, saline, citrate, phosphate, phosphate buffered saline (PBS), acetate, glycine, tris(hydroxymethyl)aminomethane (tris)hydrochloride Salts, Tris-buffered saline (TBS), 3-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]propane-1-sulfonic acid (TAPS), bicine, tricine, 3 -[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]-2-hydroxypropane-1-sulfonic acid (TAPSO), 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid (HEPES), piperazine-N, N′-bis(2-ethanesulfonic acid) (PIPES), 3-(N-morpholino)propanesulfonic acid (MOPS), 2-(N-morpholino)ethanesulfonic acid ( MES), 2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid (TES), cacodylate, glycine, carbonate, or any combination thereof could be. Buffers are 0.1 mM to about 500, 0.1 mM to about 400 mM, 0.1 mM to about 300 mM, 0.1 mM to about 200 mM, 0.1 mM to about 100 mM, 0.1 mM to about 50 mM, 0.1 mM to about about 25 mM, 0.1 mM to about 20 mM, 0.1 mM to about 15 mM, 0.1 mM to about 10 mM, 0.1 mM to about 5 mM, 0.1 mM to about 4 mM, 0.1 mM to about 3 mM, 0.1 mM to about 2 mM, 0.1 mM to about 1 mM, 0.1 mM to about 0.9 mM, 0.1 mM to about 0.8 mM, 0.1 mM to about 0.7 mM, 0.1 mM to about 0.6 mM, 0.1 mM to about It can be present at a concentration of 0.5 mM, 0.1 mM to about 0.4 mM, 0.1 mM to about 0.3 mM, or 0.1 mM to about 0.2 mM. Buffers are less than 500 mM, less than 400 mM, less than 300 mM, less than 200 mM, less than 100 mM, less than 50 mM, less than 25 mM, less than 20 mM, less than 15 mM, less than 10 mM, less than 5 mM, less than 4 mM, less than 3 mM, less than 2 mM, less than 1 mM, 0 It may be present at a concentration of less than 0.9 mM, less than 0.8 mM, less than 0.7 mM, less than 0.6 mM, less than 0.5 mM, less than 0.4 mM, less than 0.3 mM, less than 0.2 mM, or 0.1 mM. Buffers can be present at about 0.1 mM, 1 mM, 10 mM, 25 mM or 50 mM. Buffers can be present at at least 0.1 mM, 1 mM, 10 mM, 25 mM, or 50 mM.

[0143] The one or more surfactants can be, for example, anionic, cationic, nonionic or amphoteric. The one or more surfactants are, for example, polyethoxylated alcohols, polyoxyethylene sorbitan, octoxynol (polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ethers), polysorbates such as Tween™ 20 (e.g. polysorbate 20) or Tween™ 80 (polysorbate 80), sodium dodecyl sulfate, sodium lauryl sulfate, nonylphenol ethoxylates such as Tergitol™, cyclodextrins, or It can be any combination of these. Each of the one or more surfactants is less than 0.001 vol.%, less than 0.005 vol.%, less than 0.01 vol.%, less than 0.015 vol.%, 0 less than 0.02% by volume, less than 0.025% by volume, less than 0.03% by volume, less than 0.035% by volume, less than 0.04% by volume, less than 0.045% by volume, less than 0.05% by volume; 055% by volume, less than 0.06% by volume, less than 0.065% by volume, less than 0.07% by volume, less than 0.075% by volume, less than 0.08% by volume, less than 0.085%, 0.09% by volume Less than, less than 0.095% by volume, less than 0.1% by volume, less than 0.15% by volume, less than 0.2% by volume, less than 0.25% by volume, less than 0.3% by volume, less than 0.35% by volume , less than 0.4% by volume, less than 0.45% by volume, less than 0.5% by volume, less than 0.55% by volume, less than 0.6% by volume, less than 0.65% by volume, less than 0.7% by volume; Less than 0.75% by volume, less than 0.8% by volume, less than 0.85% by volume, less than 0.9% by volume, less than 0.95% by volume, less than 0.1% by volume, less than 1% by volume, 2% by volume may be present at a concentration of less than or less than 3% by volume. One or more surfactants may be present at a concentration of about 0.01%, 0.05%, 0.1%, 0.5%, 1%, 5% or 10%. One or more surfactants may be present at a concentration of at least about 0.01%, 0.05%, 0.1%, 0.5%, 1%, 5% or 10%. one or more surfactants, about 0.01%-1%, about 0.05%-1%, about 0.1%-1%, about 0.15%-1%, about 0.2% ~1%, about 0.25%-1%, about 0.3%-1%, about 0.35%-1%, about 0.4%-1%, about 0.45%-1%, about 0.5%-1%, about 0.55%-1%, about 0.6%-1%, about 0.65%-1%, about 0.7%-1%, about 0.75%- It can be present at a concentration of 1%, about 0.8%-1%, about 0.85%-1%, about 0.9%-1%, or about 0.95%-1%.

[0144] The elution buffer is about 2 to about 10, about 2 to about 9, about 2 to about 8, about 2 to about 7, about 2 to about 6, about 2 to about 5, about 2 to about 4, Or it can be an aqueous solution having a pH of about 2 to about 3. Elution buffers are about 6 to about 8, about 6 to about 7.9, about 6 to about 7.8, about 6 to about 7.7, about 6 to about 7.6, about 6 to about 7.5 , about 6 to about 7.4, about 6 to about 7.3, about 6 to about 7.2, about 6 to about 7.1, about 6 to about 7, about 6 to about 6.9, about 6 to about 6.8, about 6 to about 6.7, about 6 to about 6.6, about 6 to about 6.5, about 6 to about 6.4, about 6 to about 6.3, about 6 to about 6 .2, or an aqueous solution having a pH of from about 6 to about 6.1. The elution buffer can be an aqueous solution having a pH of at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 5, at least about 8, at least about 9, or at least about 10. The pH can be about 6, 6.5, 7, 7.5, 8 or 8.5.

[0145] Optionally, the one or more polyols are glycols such as ethylene glycol or propylene glycol, or glycol polymers such as polyethylene glycols (PEG) of various molecular weights such as PEG300, PEG400, PEG600, PEG1000, PEG3000 and PEG6000. can be In some cases, one or more polyols can be sugars. In some cases, the sugar can be sucrose, glucose, fructose, trehalose, maltose, galactose, lactose, or any combination thereof. In some cases, one or more polyols can be sugar alcohols. Optionally, the sugar alcohol can be glycerol, erythritol, threitol, xylitol, sorbitol, and the like.

[0146] The one or more salts are sodium chloride, sodium acetate, sodium bicarbonate, sodium bisulfate, sodium bromide, potassium chloride, potassium acetate, potassium bicarbonate, potassium bisulfate, potassium bromate, potassium bromide, or potassium carbonate. One or more salts can be at a concentration of about 0.1 mM, 5 mM, 10 mM, 25 mM, 50 mM, 100 mM, 250 mM, 500 mM, or 750 mM. One or more salts can be at concentrations less than 0.1 mM, 5 mM, 10 mM, 25 mM, 50 mM, 100 mM, 250 mM, 500 mM, or 750 mM. One or more salts can be at a concentration of at least 0.1 mM, 5 mM, 10 mM, 25 mM, 50 mM, 100 mM, 250 mM, 500 mM, 750 mM or 1000 mM.

[0147] The one or more blocking agents can be bovine serum albumin, fetal bovine serum, and the like.

[0148] The one or more organic solvents can be, for example, methanol, ethanol, DMSO, DMF, dioxane, tetrahydrofuran, propanol, isopropanol, butanol, t-butanol or pentanol, acetone, and the like. The elution buffer is less than 0.01 vol.%, less than 0.05 vol.%, less than 0.1 vol.%, less than 0.15 vol.%, less than 0.2 vol.%, 0.01 vol.%, less than 0.05 vol.%, less than 0.2 vol. Less than 25% by volume, less than 0.3% by volume, less than 0.35% by volume, less than 0.4% by volume, less than 0.45% by volume, less than 0.5% by volume, less than 0.55% by volume, 0.6 Less than 0.65% by volume, less than 0.7% by volume, less than 0.75% by volume, less than 0.8% by volume, less than 0.85% by volume, less than 0.9% by volume, 0.95% by volume %, less than 1 vol.%, less than 1.5 vol.%, less than 2 vol.%, less than 2.5 vol.%, less than 3 vol.%, less than 3.5 vol.%, less than 4 vol.%, less than 4.5 vol.% , less than 5% by volume, less than 5.5% by volume, less than 6% by volume, less than 6.5% by volume, less than 7% by volume, less than 7.5% by volume, less than 8% by volume, less than 8.5% by volume, 9 Less than 9.5% by volume, less than 10% by volume, less than 11% by volume, less than 12% by volume, less than 13% by volume, less than 14% by volume, less than 15% by volume, less than 16% by volume, less than 17% by volume , less than 18% by volume, less than 19% by volume, less than 20% by volume, less than 25% by volume, less than 30% by volume, less than 35% by volume, less than 40% by volume, less than 45% by volume, less than 50% by volume, less than 55% by volume , less than 60% by volume, less than 65% by volume, less than 70% by volume, less than 75% by volume, less than 80% by volume, less than 85% by volume, less than 90% by volume, less than 95% by volume, less than 99% by volume, or 100% by volume may contain less than organic solvent. The concentration of one or more organic solvents in the elution buffer can be at least 1%, 5%, 10%, 50%, 75%, or 100%. The concentration of one or more organic solvents in the elution buffer can be about 1%, 5%, 10%, 50%, 75%, or 100%.

[0149] The stabilizing matrix or a portion of the stabilizing matrix is about or <60 μL, <65 μL, <70 μL, <75 μL, <80 μL, <85 μL, <90 μL, <95 μL, <100 μL, <110 μL, <120 μL, <130 μL, <140 μL, <150 μL, <160 μL, <170 μL, <180 μL , <190 μL, <200 μL, <250 μL, <300 μL, <350 μL, <400 μL, <450 μL, <500 μL, <550 μL, <600 μL, <650 μL, <700 μL, <750 μL, <800 μL, <850 μL, <900 μL, 950 μL Less than, less than 1,000 μL, less than 1.5 mL, less than 2 mL, less than 2.5 mL, less than 3 mL, less than 3.5 mL, less than 4 mL, less than 4.5 mL, less than 5 mL, less than 5.5 mL, less than 6 mL, 6.5 mL Less than, less than 7 mL, less than 7.5 mL, less than 8 mL, less than 8.5 mL, less than 9 mL, less than 9.5 mL, or less than 10 mL of elution buffer volume can be contacted. The stabilizing matrix or a portion of the stabilizing matrix can be contacted with about 0.1 mL, 0.2 mL, 0.5 mL, 0.7 mL, 1 mL, 2 mL, 5 mL, 7 mL or 10 mL or more of elution buffer. can. The stabilizing matrix or a portion of the stabilizing matrix can be contacted with at least 0.1 mL, 0.2 mL, 0.5 mL, 0.7 mL, 1 mL, 2 mL, 5 mL, 7 mL or 10 mL of elution buffer.

[0150] The volume of elution buffer that contacts the stabilizing matrix may depend on the surface area of the stabilizing matrix. The amount of elution buffer is less than 1 μL/mm2, less than ² μL/ ^mm2 , less than 3 μL/ ^mm2 , less than 4 μL/ ^mm2 , less than 5 μL/ ^mm2 , less than 6 μL/ ^mm2 , less than 7 μL/ ^mm2 , 8 μL. / ^mm2 , less than 9 μL/ ^mm2 , less than 10 μL/ ^mm2 , less than 12 μL/ ^mm2 , less than 14 μL/ ^mm2 , less than 16 μL/ ^mm2 , less than 18 μL/ ^mm2 , less than 20 μL/ ^mm2 , 25 μL/mm2 less than ² , less than 30 μL/ ^mm2 , less than 35 μL/ ^mm2 , less than 40 μL/ ^mm2 , less than 45 μL/ ^mm2 , less than 50 μL/ ^mm2 , less than 55 μL/ ^mm2 , less than 60 μL/ ^mm2 , less than 65 μL/ ^mm2 , less than 70 μL/ ^mm2 , less than 75 μL/ ^mm2 , less than 80 μL/ ^mm2 , less than 85 μL/ ^mm2 , less than 90 μL/ ^mm2 , less than 95 μL/ ^mm2 , less than 100 μL/ ^mm2 , less than 150 μL/ ^mm2 , 200 μL / ^mm2 , less than 250 μL/ ^mm2 , less than 300 μL/ ^mm2 , less than 350 μL/ ^mm2 , less than 400 μL/ ^mm2 , less than 450 μL/ ^mm2 , less than 500 μL/ ^mm2 , less than 550 μL/ ^mm2 , 600 μL/mm2 less than ² , less than 650 μL/ ^mm2 , less than 700 μL/ ^mm2 , less than 750 μL/ ^mm2 , less than 800 μL/ ^mm2 , less than 850 μL/ ^mm2 , less than 900 μL/ ^mm2 , less than 950 μL/ ^mm2 , or 1,000 μL/mm2 may be less than ^mm2 . The amount of elution buffer is about 10 μL/mm ² to about 1,000 μL/mm ² , about 10 μL/mm ² to about 900 μL/mm ² , about 10 μL/mm ² to about 800 μL/mm ² , about 10 μL/mm ² . to about 700 μL/mm ² , about 10 μL/mm ² to about 600 μL/mm ² , about 10 μL/mm ² to about 500 μL/mm ² , about 10 μL/mm ² to about 400 μL/mm ² , about 10 μL/mm ² to about 300 μL/mm ² , about 10 μL/mm ² to about 200 μL/mm ² , about 10 μL/mm ² to about 100 μL/mm ² , about 10 μL/mm ² to about 90 μL/mm ² , about 10 μL/mm ² to about 80 μL/mm 2 . mm ² , about 10 μL/mm ² to about 70 μL/mm ² , about 10 μL/mm ² to about 60 μL/mm ² , about 10 μL/mm ² to about 50 μL/mm ² , about 10 μL/mm ² to about 40 μL/mm ² , from about 10 μL/mm ² to about 30 μL/mm ² , or from about 10 μL/mm ² to about 20 μL/mm ² .

[0151] Proteins can be eluted from the stabilization matrix by incubating the stabilization matrix in an elution buffer with or without agitation from a source of agitation. Agitation sources can be rockers, vortex mixers, mixers, shakers, and the like. In some cases, the agitation source can be set at a constant speed. The speed is less than 1 revolution per minute (rpm), less than 5 rpm, less than 10 rpm, less than 15 rpm, less than 20 rpm, less than 25 rpm, less than 30 rpm, less than 35 rpm, less than 40 rpm, less than 45 rpm, less than 50 rpm, less than 55 rpm, less than 60 rpm, less than 65 rpm , less than 70 rpm, less than 75 rpm, less than 80 rpm, less than 85 rpm, less than 90 rpm, less than 95 rpm, less than 100 rpm, less than 150 rpm, less than 200 rpm, less than 250 rpm, less than 300 rpm, less than 350 rpm, less than 400 rpm, less than 450 rpm, less than 500 rpm, less than 550 rpm, 600 rpm less than, less than 650 rpm, less than 700 rpm, less than 750 rpm, less than 800 rpm, less than 850 rpm, less than 900 rpm, less than 950 rpm, less than 1,000 rpm, less than 1,500 rpm, less than 2,000 rpm, less than 2,500 rpm, less than 3,000 rpm, 3, less than 500 rpm, less than 4,000 rpm, less than 4,500 rpm, less than 5,000 rpm, less than 5,500 rpm, less than 6,000 rpm, less than 6,500 rpm, less than 7,000 rpm, less than 7,500 rpm, less than 8,000 rpm; It can be less than 500 rpm, less than 9,000 rpm, less than 9,500 rpm, or less than 10,000 rpm. The speed can be about 50 rpm, 100 rpm, 200 rpm, 300 rpm, 400 rpm, 500 rpm, 600 rpm, 700 rpm, 800 rpm, 900 rpm, 1000 rpm, 1500 rpm or 5000 rpm. The speed can be at least 50 rpm, 100 rpm, 200 rpm, 300 rpm, 400 rpm, 500 rpm, 600 rpm, 700 rpm, 800 rpm, 900 rpm, 1000 rpm, 1500 rpm or 5000 rpm.

[0152] Elution is at about 0°C or less, 1°C or less, 2°C or less, 3°C or less, 4°C or less, 5°C or less, 6°C or less, 7°C or less, 8°C. less than, less than 9°C, less than 10°C, less than 11°C, less than 12°C, less than 13°C, less than 14°C, less than 15°C, less than 16°C, less than 17°C, 18°C less than, less than 19°C, less than 20°C, less than 21°C, less than 22°C, less than 23°C, less than 24°C, less than 25°C, less than 26°C, less than 27°C, 28°C less than, less than 29°C, less than 30°C, less than 31°C, less than 32°C, less than 33°C, less than 34°C, less than 35°C, less than 36°C, less than 37°C, 38°C less than, less than 39°C, less than 40°C, less than 45°C, less than 50°C, less than 55°C, less than 60°C, less than 65°C, less than 70°C, less than 75°C, 80°C can be carried out at temperatures below, below 85°C, below 90°C, below 95°C, or about 100°C. Elution is from about 10° C. to about 100° C., from about 10° C. to about 95° C., from about 10° C. to about 90° C., from about 10° C. to about 85° C., from about 10° C. to about 80° C. °C, about 10°C to about 75°C, about 10°C to about 70°C, about 10°C to about 65°C, about 10°C to about 60°C, about 10°C to about 55°C °C, about 10°C to about 50°C, about 20°C to about 50°C, about 20°C to about 48°C, about 20°C to about 46°C, about 20°C to about 44°C °C, about 20°C to about 42°C, about 20°C to about 40°C, about 20°C to about 38°C, about 20°C to about 36°C, about 20°C to about 34°C °C, about 20°C to about 32°C, about 20°C to about 30°C, about 20°C to about 28°C, about 20°C to about 26°C, about 20°C to about 24°C °C, or at temperatures from about 20°C to about 22°C. Elution can be performed at about 10°C, 20°C, 25°C, 30°C, 37°C, 50°C, or 65°C.

[0153] Elution (e.g., agitation) takes less than 1 minute, less than 5 minutes, less than 10 minutes, less than 15 minutes, less than 20 minutes, less than 25 minutes, less than 30 minutes, less than 35 minutes, less than 40 minutes, less than 45 minutes. , less than 50 minutes, less than 55 minutes, or less than 60 minutes. Elution was less than 0.5 hours, less than 1 hour, less than 1.5 hours, less than 2 hours, less than 2.5 hours, less than 3 hours, less than 3.5 hours, less than 4 hours, less than 4.5 hours, 5 hours, less than 5.5 hours, less than 6 hours, less than 6.5 hours, less than 7 hours, less than 7.5 hours, less than 8 hours, less than 8.5 hours, less than 9 hours, less than 9.5 hours, 10 can be conducted for less than 1 hour, less than 10.5 hours, less than 11 hours, less than 11.5 hours, or less than 12 hours. Elution less than 0.1 days, less than 0.2 days, less than 0.3 days, less than 0.4 days, less than 0.5 days, less than 0.6 days, less than 0.7 days, less than 0.8 days , less than 0.9 days, less than 1 day, less than 1.5 days, less than 2 days, less than 2.5 days, less than 3 days, less than 3.5 days, less than 4 days, less than 4.5 days, less than 5 days , less than 5.5 days, less than 6 days, less than 6.5 days, or less than 7 days. Elution (e.g., agitation) is performed for about 0.25 hours, 0.5 hours, 1 hour, 2 hours, 5 hours, 10 hours, 12 hours, 18 hours, 24 hours, 3 days, or 1 week. can do. Elution (e.g., agitation) is performed for at least 0.25 hours, 0.5 hours, 1 hour, 2 hours, 5 hours, 10 hours, 12 hours, 18 hours, 24 hours, 3 days, or 1 week. can do. Table 1 lists examples of protein elution buffers.

[0154]

[0155] The eluted protein can be transferred to a container for storage or further processing, or can be transferred to an assay container for characterization.

[0156] USE OF MATRIX

[0157] The sample can be applied to one matrix (eg, one layer). A sample can be applied to the top matrix and at least a portion of the sample can be in contact with a second matrix, eg, a second matrix underlying the top matrix. The matrix can be stacked in, for example, about or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or 100 layers. A stack can include the same matrix, eg, each matrix within a stack can have the same composition. A stack can include matrices having different compositions, for example, a matrix configured for stabilizing nucleic acids can be positioned on top of a matrix configured for stabilizing proteins. , and vice versa. The types of matrices within the stack alternate, e.g., matrices configured to stabilize proteins, matrices configured to stabilize nucleic acids, followed by matrices configured to stabilize proteins. followed by the matrix. The volume applied to the top matrix can pass through at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 layers of matrix. The use of multiple matrices can increase recovery of desired biomolecules, such as nucleic acids or polypeptides.

[0158] The sample can be passed through multiple matrices, but the matrices do not contact each other. For example, the sample can be applied to a matrix configured to stabilize nucleic acids, the nucleic acids are eluted from the matrix configured to stabilize nucleic acids, and the It can be applied to matrices. Matrices configured to stabilize proteins can be used, for example, to remove contaminants, such as protein contaminants, from nucleic acids. The sample can be applied to a matrix configured to stabilize proteins, eluting the proteins from the matrix configured to stabilize proteins, and applying the sample to the matrix configured to stabilize nucleic acids. can be applied. Matrices configured to stabilize nucleic acids can be used, for example, to remove contaminants, such as nucleic acid contaminants, from proteins.

[0159] Embodiment

[0160] Example 1: Methods and Kits for Tagged Sample Collection and Preparation

[0161] A sample acquisition and stabilization component is deployed to the end user. A sample stabilization component is specifically configured for a specified diagnostic test set and is specifically configured for a particular target component. For example, DNA, RNA and protein target components each have different substrate compositions and distinguishing colors. Tests configured for the RNA target component have a red stabilization matrix, tests configured for DNA have a green stabilization matrix, and tests configured for the protein target component have a red stabilization matrix. It has a blue stabilizing matrix. In addition, each deployed system has a unique barcode for batch processing samples and for linking those sample results with identification information corresponding to the donor from which the samples originated. Second, it is used to sort and separate samples by type (RNA/DNA/protein). The sample stabilizing component has a plurality of barcode labels, one of which is a label that remains permanently affixed to the sample stabilizing component, some of which are easy to process. It is a removable adhesive label. The label displays a unique bar code associated with the deployed system. After acquiring a sample using the sample acquisition and stabilization system, the system is returned to the facility where it was collected. Fixed barcodes are used to mechanically separate incoming systems by board type. For example, all sample stabilization components with a red stabilization matrix are collected together and assembled into batches of 96. Assays are performed based on the color tag of the samples. A batch of 96 red samples are collected together and two labels from each sample are transferred to two sets of 96 RNAse-free tubes, each assembled sequentially based on the order in which they are scanned. Each rack of 96 labeled sample tubes is set up and structured identically. Open the sample stabilization component of each of the 96 samples in scan order, remove the red substrate, and place it like a filter on the rim placed in the RNAse-free sample tube with the corresponding sample barcode label. do. This is done until the substrate of each of the 96 samples is correctly labeled and placed in the corresponding 96 sample tubes. Opening the "red kit" designed for target components from the red substrate reveals a red-capped container containing RNase-free PCR buffers, reagents and other molecular components. The bottles also have step numbers on them so that they can be used simply and quickly to perform different treatment steps on the red samples. Add an aliquot of "Step 1 Red Reagent" to the top of the red substrate in each of the 96 labeled tubes, close the tube, and allow the reagent to sit for a few minutes to soak the substrate. After immersion in reagents and buffers, the sample is centrifuged to force the contents of the substrate into the liquid solution formed at the bottom of the sample tube. Another aliquot of "Step 1 Red Reagent" is added again on top of the substrate and centrifugation is repeated. A tube is opened and a solid substrate is removed from each sample, followed by a buffer solution containing partially double-stranded DNA molecules with single-stranded regions complementary to the target component RNA ("Step 2 Red Add an aliquot of "reagent" to the liquid solution. The tube is closed and placed in a PCR machine at a temperature that promotes Brownian motion without inducing denaturation of the double-stranded DNA molecules. At this temperature, RNA from the substrate hybridizes to single-stranded regions of DNA molecules. The DNA molecule has a promoter for RNA polymerase within the double-stranded region and the 3' overhang of the single-stranded region has a strand of thymidine residues. A poly(A) tail at the 3' end of the messenger RNA (mRNA) from the red substrate hybridizes with the thymine residue overhangs of the DNA molecule. The tube is then opened and an aliquot of "Step 3 Red Reagent" containing ligase enzyme and buffer is added to the tube. Upon heating the sample, ligation occurs between the mRNA and the double-stranded DNA molecule, forming a double-stranded RNA-DNA molecule, with the entire mRNA incorporated as one strand of the molecule. opening the tubes, adding an aliquot of "Step 4 Red Reagent" containing RNA polymerase to each of the 96 tubes, closing the tubes, and performing 32 cycles of PCR with repeated denaturation and annealing on the samples; An RNA template is amplified to generate a library of antisense cRNAs. Transfer an aliquot of amplified cRNA PCR product to each of the corresponding 96 empty labeled sample tubes and transfer an aliquot of "Step 1 Red Reagent" to a second rack of 96 empty labeled tubes. add to Steps 2-4 are repeated to perform ligation and polymerization, which now yields the sense strand of mRNA. These sense strands were then sequenced using standard sequencing protocols and the results analyzed using standard gene expression profiling methods, using barcode numbers as identifiers, resulting in Determine the donor associated with the outcome.

[0162] Other kits are also available for performing similar batch assays. The green kit is for green substrate components that selectively stabilize DNA, and the blue kit is for blue substrate components that selectively stabilize proteins. Different methods are used to treat samples from each of these different substrates.

[0163] Example 2: Kit with Lancet and Tourniquet with Sample Separation Components

[0164] The kit is deployed to the end user or donor. The kit includes crystal-activated warmers for hand warming, lancets, tourniquets, alcohol pads, gauze, pressure-activated lancets, self-filling capillaries, and sample stabilization with an integrated sample separation unit and sample stabilization matrix. contains a conversion component. The end user warms the donor's hand to facilitate stimulation of blood flow prior to puncturing. This is accomplished by activating a crystal-activated handwarmer warmer and holding it between the fingers. The donor's hands are relaxed and positioned under the heart and muscles while the donor sits comfortably in the chair with the hands and arms loosely placed on the arms of the chair. A tourniquet is placed on the donor's non-dominant hand to select a site on the donor's middle finger. A rubber band tourniquet is wrapped around the last finger and then twisted a few more times until it loops around the finger to form a tourniquet. Leave the loop for easy retrieval. Pressure is applied to the fingertips, causing them to become slightly red and raised. Sterilize the sample site. First, the side of the fingertip is selected and a piece of sterile gauze, alcohol pad wipe is performed through the area before it dries. The donor holds and pulls on the loose loop of the tourniquet during the puncture. The lancing process may depend on the type and source of lancet provided. Remove the lancet's protective cap and position the lancet toward the sterile finger side. The center of the fingertip is stiffened and contains a higher density of nerve endings, so place the lancet away from the center of the fingertip. The lancet is pushed down until the spring within the lancet engages and an audible click is heard, indicating that the skin has been punctured. The first blood after the puncture is removed immediately and light but constant pressure is applied to the finger. Hold the self-filling capillary horizontal to the incision site and contact the blood drop that forms using a self-filling capillary (eg, Microsafe®, Safe-Tec Clinical Products, LLC, Ivyland PA). Let The capillary will self-fill and self-stop to the black line printed on the plastic shaft. Due to the presence of the plastic spheres, they cannot fall during the filling step. When the collected blood reaches the black line and the filling is complete, the pressure is released from the fingertip and the free loop of the rubber band is released to reduce pressure on the finger tourniquet. Blood is dispensed into the sample stabilizing component. Place the sample stabilization component on a flat surface and wipe the blood outside the capillary clean with sterile gauze. The filled capillary is held upright on the bottom of the sample stabilization component and the collected sample is dispensed by gently and evenly pressing the plastic sphere of the filled capillary. The capillary is fixed in place at the bottom of the sample stabilization component during dispensing. The capillary is discarded when all blood has been dispensed onto the sample stabilization component. After the procedure, the blood sampling component is allowed to rest while the finger tourniquet is completely removed and the incision site is cleaned. Apply pressure to the incision with sterile gauze to stop bleeding and raise your hand above the heart to aid in clotting. The sample stabilization component is allowed to sit for approximately 5-10 minutes after the procedure and observed to determine if the blood droplets are still heaving on the filter and if the filter still looks "wet." In this case, using the separation component, a raised "wet" drop of blood is observed and then wheat-colored plasma begins to appear on top of the sample separation component. The appearance of sample separation is used to indicate that the sample can be returned to the reservoir. Blood sampling components are labeled with barcode labels and allowed to stand at room temperature. Seal the storage container and place it in the mail.

[0165] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, modifications, and substitutions can be made by those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be used in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered therein.

Claims (26)

A method of preserving blood collected from a subject, comprising:
(a) obtaining an integrated sample acquisition and sample stabilization device, said device comprising: (i) a sample comprising one or more piercing elements configured to pierce the skin and expose blood; an acquisition component; (ii) a plurality of paper sample stabilization matrices for collecting and storing said blood, said plurality of paper sample stabilization matrices stacked with one or more layers interposed; and the plurality of paper sample stabilization matrices do not contact each other;
(b) moving said blood from an opening of said sample acquisition component through a channel and onto a plurality of said paper sample stabilization matrices that are stacked and do not contact each other, thereby preserving said blood;
method including. 2. The method of claim 1, wherein a vacuum causes the blood to move from the opening of the sample acquisition component through the channel and onto a plurality of paper sample stabilization matrices that are stacked and do not contact each other. 3. The method of claim 1 or 2, wherein step ( b ) comprises moving more than 100 [mu]l of said blood. 4. The method of any one of claims 1-3, wherein the one or more piercing elements comprises a plurality of piercing elements. 5. The method of any one of claims 1-4, wherein the one or more lancing elements are lancets. 5. The method of any one of claims 1-4, wherein the one or more piercing elements are solid. 7. The method of any one of claims 1-6, wherein the blood is capillary blood. 8. The method of any one of claims 1-7, wherein the device is a disposable device. 9. The method of any one of claims 1-8, wherein the plurality of paper sample stabilization matrices comprises cellulose, cellulose acetate, glass fibers, or combinations thereof. 10. The method of any one of claims 1-9, wherein the blood collected by the combined sample acquisition and sample stabilization device comprises a volume of less than 500 [mu]L. 11. The method of any one of claims 1-10, further comprising removing the plurality of paper sample stabilization matrices containing the blood from the combined sample acquisition and sample stabilization device. 12. The method of claim 11, further comprising performing an assay on blood from at least one of said plurality of paper sample stabilization matrices containing said blood. 13. The method of claim 12, further comprising eluting DNA, RNA, or protein in the blood from at least one of the plurality of paper sample stabilization matrices containing the blood. 14. The method of claim 12 or 13, wherein said assay is performed at a different location than where said blood is collected using said device. 15. The method of any one of claims 1-14, wherein the channel comprises a microchannel. Moving blood from the opening of the sample acquisition component, through the channels, and onto the plurality of paper sample stabilization matrices can be accomplished by naturally occurring capillary forces, wicking through an absorbent material, or a combination thereof. 3. The method of claim 2, also caused by 17. The method of any one of claims 1-16, wherein the one or more piercing elements are connected to a spring. 18. The method of any one of claims 1-17, wherein the blood is absorbed by the plurality of paper sample stabilization matrices. 19. The method of any one of claims 1-18, wherein the plurality of paper sample stabilization matrices are removable from the device. 20. The method of any one of claims 1-19, wherein the plurality of paper sample stabilization matrices are disposable. 21. The method of any one of claims 1-20, further comprising storing the plurality of blood-containing paper sample stabilization matrices of step b) at a temperature in the range of 15°C to 40°C. . 22. The method of any one of claims 1-21, further comprising storing the plurality of paper sample stabilization matrices containing the blood of step b) for at least one week. 23. The method of any one of claims 1-22, wherein the plurality of paper sample stabilization matrices comprises paper cards. Moving blood from the opening of the sample acquisition component, through the channels, and onto the plurality of paper sample stabilization matrices can be accomplished by naturally occurring capillary forces, wicking through an absorbent material, or a combination thereof. A method according to any one of claims 1, 3-23, produced by The plurality of paper sample stabilizing matrices comprises one or more stabilizing reagents, the one or more stabilizing reagents comprising a trisaccharide, the plurality of paper sample stabilizing matrices comprising one or more organisms. 2. The method of claim 1, configured to selectively stabilize biological components, wherein said one or more biological components are proteins. 2. The method of claim 1, wherein said plurality of paper sample stabilization matrices are in a substantially dry state with less than 10% hydration by weight.

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